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Viruses, Vol. 10, Pages 662: The Transcriptional Repressor BS69 is a Conserved Target of the E1A Proteins from Several Human Adenovirus Specieshttp://www.mdpi.com/1999-4915/10/12/662
Early region 1A (E1A) is the first viral protein produced upon human adenovirus (HAdV) infection. This multifunctional protein transcriptionally activates other HAdV early genes and reprograms gene expression in host cells to support productive infection. E1A functions by interacting with key cellular regulatory proteins through short linear motifs (SLiMs). In this study, the molecular determinants of interaction between E1A and BS69, a cellular repressor that negatively regulates E1A transactivation, were systematically defined by mutagenesis experiments. We found that a minimal sequence comprised of MPNLVPEV, which contains a conserved PXLXP motif and spans residues 112&amp;ndash;119 in HAdV-C5 E1A, was necessary and sufficient in binding to the myeloid, Nervy, and DEAF-1 (MYND) domain of BS69. Our study also identified residues P113 and L115 as critical for this interaction. Furthermore, the HAdV-C5 and -A12 E1A proteins from species C and A bound BS69, but those of HAdV-B3, -E4, -D9, -F40, and -G52 from species B, E, D, F, and G, respectively, did not. In addition, BS69 functioned as a repressor of E1A-mediated transactivation, but only for HAdV-C5 and HAdV-A12 E1A. Thus, the PXLXP motif present in a subset of HAdV E1A proteins confers interaction with BS69, which serves as a negative regulator of E1A mediated transcriptional activation.Viruses, Vol. 10, Pages 662: The Transcriptional Repressor BS69 is a Conserved Target of the E1A Proteins from Several Human Adenovirus Species

Early region 1A (E1A) is the first viral protein produced upon human adenovirus (HAdV) infection. This multifunctional protein transcriptionally activates other HAdV early genes and reprograms gene expression in host cells to support productive infection. E1A functions by interacting with key cellular regulatory proteins through short linear motifs (SLiMs). In this study, the molecular determinants of interaction between E1A and BS69, a cellular repressor that negatively regulates E1A transactivation, were systematically defined by mutagenesis experiments. We found that a minimal sequence comprised of MPNLVPEV, which contains a conserved PXLXP motif and spans residues 112&amp;ndash;119 in HAdV-C5 E1A, was necessary and sufficient in binding to the myeloid, Nervy, and DEAF-1 (MYND) domain of BS69. Our study also identified residues P113 and L115 as critical for this interaction. Furthermore, the HAdV-C5 and -A12 E1A proteins from species C and A bound BS69, but those of HAdV-B3, -E4, -D9, -F40, and -G52 from species B, E, D, F, and G, respectively, did not. In addition, BS69 functioned as a repressor of E1A-mediated transactivation, but only for HAdV-C5 and HAdV-A12 E1A. Thus, the PXLXP motif present in a subset of HAdV E1A proteins confers interaction with BS69, which serves as a negative regulator of E1A mediated transcriptional activation.

]]>The Transcriptional Repressor BS69 is a Conserved Target of the E1A Proteins from Several Human Adenovirus SpeciesAli ZhangTanner M. TessierKristianne J. C. GalpinCason R. KingSteven F. GameiroWyatt W. AndersonAhmed F. YousefWen T. QinShawn S. C. LiJoe S. Mymrykdoi: 10.3390/v10120662Viruses2018-11-22Viruses2018-11-221012Article66210.3390/v10120662http://www.mdpi.com/1999-4915/10/12/662Viruses, Vol. 10, Pages 661: ZIKV Demonstrates Minimal Pathologic Effects and Mosquito Infectivity in Viremic Cynomolgus Macaqueshttp://www.mdpi.com/1999-4915/10/11/661
To evaluate the effects of ZIKV infection on non-human primates (NHPs), as well as to investigate whether these NHPs develop sufficient viremia to infect the major urban vector mosquito, Aedes aegypti, four cynomolgus macaques (Macaca fascicularis) were subcutaneously infected with 5.0 log10 focus-forming units (FFU) of DNA clone-derived ZIKV strain FSS13025 (Asian lineage, Cambodia, 2010). Following infection, the animals were sampled (blood, urine, tears, and saliva), underwent daily health monitoring, and were exposed to Ae. aegypti at specified time points. All four animals developed viremia, which peaked 3&amp;ndash;4 days post-infection at a maximum value of 6.9 log10 genome copies/mL. No virus was detected in urine, tears, or saliva. Infection by ZIKV caused minimal overt disease: serum biochemistry and CBC values largely fell within the normal ranges, and cytokine elevations were minimal. Strikingly, the minimally colonized population of Ae. aegypti exposed to viremic animals demonstrated a maximum infection rate of 26% during peak viremia, with two of the four macaques failing to infect a single mosquito at any time point. These data indicate that cynomolgus macaques may be an effective model for ZIKV infection of humans and highlights the relative refractoriness of Ae. aegypti for ZIKV infection at the levels of viremia observed.Viruses, Vol. 10, Pages 661: ZIKV Demonstrates Minimal Pathologic Effects and Mosquito Infectivity in Viremic Cynomolgus Macaques

To evaluate the effects of ZIKV infection on non-human primates (NHPs), as well as to investigate whether these NHPs develop sufficient viremia to infect the major urban vector mosquito, Aedes aegypti, four cynomolgus macaques (Macaca fascicularis) were subcutaneously infected with 5.0 log10 focus-forming units (FFU) of DNA clone-derived ZIKV strain FSS13025 (Asian lineage, Cambodia, 2010). Following infection, the animals were sampled (blood, urine, tears, and saliva), underwent daily health monitoring, and were exposed to Ae. aegypti at specified time points. All four animals developed viremia, which peaked 3&amp;ndash;4 days post-infection at a maximum value of 6.9 log10 genome copies/mL. No virus was detected in urine, tears, or saliva. Infection by ZIKV caused minimal overt disease: serum biochemistry and CBC values largely fell within the normal ranges, and cytokine elevations were minimal. Strikingly, the minimally colonized population of Ae. aegypti exposed to viremic animals demonstrated a maximum infection rate of 26% during peak viremia, with two of the four macaques failing to infect a single mosquito at any time point. These data indicate that cynomolgus macaques may be an effective model for ZIKV infection of humans and highlights the relative refractoriness of Ae. aegypti for ZIKV infection at the levels of viremia observed.

]]>ZIKV Demonstrates Minimal Pathologic Effects and Mosquito Infectivity in Viremic Cynomolgus MacaquesSasha R. AzarShannan L. RossiSherry H. HallerRuimei YunJing H. HuangJessica A. PlanteJiehua ZhouJuan P. OlanoChristopher M. RoundyKathryn A. HanleyScott C. WeaverNikos Vasilakisdoi: 10.3390/v10110661Viruses2018-11-21Viruses2018-11-211011Article66110.3390/v10110661http://www.mdpi.com/1999-4915/10/11/661Viruses, Vol. 10, Pages 660: Non-Structural Protein NSm of Tomato Spotted Wilt Virus Is an Avirulence Factor Recognized by Resistance Genes of Tobacco and Tomato via Different Elicitor Active Siteshttp://www.mdpi.com/1999-4915/10/11/660
Tomato spotted wilt virus (TSWV) is one of the most destructive viral pathogens of plants. Recently, a single dominant gene conferring complete resistance to TSWV (RTSW) was identified in Nicotina alata and introgressed into cultivated tobacco (N. tabacum). However, whether the TSWV carries an avirulence (Avr) factor directed against RTSW remains obscure. In the present study, we identified the non-structural protein (NSm), the movement protein of TSWV, which is an RTSW-specific Avr factor, by using two different transient expression systems. Using amino acid (aa) substitution mutants, we demonstrated the ability to induce RTSW-mediated hypersensitive response (HR) of NSm is independent of its movement function. Moreover, key substitutions (C118Y and T120N), a 21-aa viral effector epitope, and different truncated versions of NSm, which are responsible for the recognition of the Sw-5b resistance gene of tomato, were tested for their ability to trigger HR to TSWV in tobacco. Together, our results demonstrated that RTSW-mediated resistance is triggered by NSm in the same way as by Sw-5b, however, via different elicitor active sites. Finally, an Avr gene-based diagnostic approach was established and used to determine the presence and effectiveness of resistance genes in tobacco.Viruses, Vol. 10, Pages 660: Non-Structural Protein NSm of Tomato Spotted Wilt Virus Is an Avirulence Factor Recognized by Resistance Genes of Tobacco and Tomato via Different Elicitor Active Sites

Tomato spotted wilt virus (TSWV) is one of the most destructive viral pathogens of plants. Recently, a single dominant gene conferring complete resistance to TSWV (RTSW) was identified in Nicotina alata and introgressed into cultivated tobacco (N. tabacum). However, whether the TSWV carries an avirulence (Avr) factor directed against RTSW remains obscure. In the present study, we identified the non-structural protein (NSm), the movement protein of TSWV, which is an RTSW-specific Avr factor, by using two different transient expression systems. Using amino acid (aa) substitution mutants, we demonstrated the ability to induce RTSW-mediated hypersensitive response (HR) of NSm is independent of its movement function. Moreover, key substitutions (C118Y and T120N), a 21-aa viral effector epitope, and different truncated versions of NSm, which are responsible for the recognition of the Sw-5b resistance gene of tomato, were tested for their ability to trigger HR to TSWV in tobacco. Together, our results demonstrated that RTSW-mediated resistance is triggered by NSm in the same way as by Sw-5b, however, via different elicitor active sites. Finally, an Avr gene-based diagnostic approach was established and used to determine the presence and effectiveness of resistance genes in tobacco.

]]>Non-Structural Protein NSm of Tomato Spotted Wilt Virus Is an Avirulence Factor Recognized by Resistance Genes of Tobacco and Tomato via Different Elicitor Active SitesChangjun HuangYong LiuHaiqin YuCheng YuanJianmin ZengLu ZhaoZhijun TongXiaorong Taodoi: 10.3390/v10110660Viruses2018-11-21Viruses2018-11-211011Article66010.3390/v10110660http://www.mdpi.com/1999-4915/10/11/660Viruses, Vol. 10, Pages 659: Understanding the Determinants of BnAb Induction in Acute HCV Infectionhttp://www.mdpi.com/1999-4915/10/11/659
Despite recent advances in curative therapy, hepatitis C virus (HCV) still remains a global threat. In order to achieve global elimination, a prophylactic vaccine should be considered high priority. Previous immunogens used to induce broad neutralising antibodies (BnAbs) have been met with limited success. To improve immunogen design, factors associated with the early development of BnAbs in natural infection must first be understood. In this study, 43 subjects identified with acute HCV were analysed longitudinally using a panel of heterogeneous HCV pseudoparticles (HCVpp), to understand the emergence of BnAbs. Compared to those infected with a single genotype, early BnAb development was associated with subjects co-infected with at least 2 HCV subtypes during acute infection. In those that were mono-infected, BnAbs were seen to emerge with increasing viral persistence. If subjects acquired a secondary infection, nAb breadth was seen to boost upon viral re-exposure. Importantly, this data highlights the potential for multivalent and prime-boost vaccine strategies to induce BnAbs against HCV in humans. However, the data also indicate that the infecting genotype may influence the development of BnAbs. Therefore, the choice of antigen will need to be carefully considered in future vaccine trials.Viruses, Vol. 10, Pages 659: Understanding the Determinants of BnAb Induction in Acute HCV Infection

Despite recent advances in curative therapy, hepatitis C virus (HCV) still remains a global threat. In order to achieve global elimination, a prophylactic vaccine should be considered high priority. Previous immunogens used to induce broad neutralising antibodies (BnAbs) have been met with limited success. To improve immunogen design, factors associated with the early development of BnAbs in natural infection must first be understood. In this study, 43 subjects identified with acute HCV were analysed longitudinally using a panel of heterogeneous HCV pseudoparticles (HCVpp), to understand the emergence of BnAbs. Compared to those infected with a single genotype, early BnAb development was associated with subjects co-infected with at least 2 HCV subtypes during acute infection. In those that were mono-infected, BnAbs were seen to emerge with increasing viral persistence. If subjects acquired a secondary infection, nAb breadth was seen to boost upon viral re-exposure. Importantly, this data highlights the potential for multivalent and prime-boost vaccine strategies to induce BnAbs against HCV in humans. However, the data also indicate that the infecting genotype may influence the development of BnAbs. Therefore, the choice of antigen will need to be carefully considered in future vaccine trials.

]]>Understanding the Determinants of BnAb Induction in Acute HCV InfectionAlexander P. UnderwoodMelanie R. WalkerNicholas A. BrasherAuda A. EltahlaLisa MaherFabio LucianiAndrew R. LloydRowena A. Bulldoi: 10.3390/v10110659Viruses2018-11-21Viruses2018-11-211011Article65910.3390/v10110659http://www.mdpi.com/1999-4915/10/11/659Viruses, Vol. 10, Pages 658: Virulence of Marburg Virus Angola Compared to Mt. Elgon (Musoke) in Macaques: A Pooled Survival Analysishttp://www.mdpi.com/1999-4915/10/11/658
Angola variant (MARV/Ang) has replaced Mt. Elgon variant Musoke isolate (MARV/MtE-Mus) as the consensus standard variant for Marburg virus research and is regarded as causing a more aggressive phenotype of disease in animal models; however, there is a dearth of published evidence supporting the higher virulence of MARV/Ang. In this retrospective study, we used data pooled from eight separate studies in nonhuman primates experimentally exposed with either 1000 pfu intramuscular (IM) MARV/Ang or MARV/MtE-Mus between 2012 and 2017 at the United States Army Medical Research Institute of Infectious Diseases (USAMRIID). Multivariable Cox proportional hazards regression was used to evaluate the association of variant type with time to death, the development of anorexia, rash, viremia, and 10 select clinical laboratory values. A total of 47 cynomolgus monkeys were included, of which 18 were exposed to MARV/Ang in three separate studies and 29 to MARV/MtE-Mus in five studies. Following universally fatal Marburg virus exposure, compared to MARV/MtE-Mus, MARV/Ang was associated with an increased risk of death (HR = 22.10; 95% CI: 7.08, 68.93), rash (HR = 5.87; 95% CI: 2.76, 12.51) and loss of appetite (HR = 35.10; 95% CI: 7.60, 162.18). Our data demonstrate an increased virulence of MARV/Ang compared to MARV/MtE-Mus variant in the 1000 pfu IM cynomolgus macaque model.Viruses, Vol. 10, Pages 658: Virulence of Marburg Virus Angola Compared to Mt. Elgon (Musoke) in Macaques: A Pooled Survival Analysis

Angola variant (MARV/Ang) has replaced Mt. Elgon variant Musoke isolate (MARV/MtE-Mus) as the consensus standard variant for Marburg virus research and is regarded as causing a more aggressive phenotype of disease in animal models; however, there is a dearth of published evidence supporting the higher virulence of MARV/Ang. In this retrospective study, we used data pooled from eight separate studies in nonhuman primates experimentally exposed with either 1000 pfu intramuscular (IM) MARV/Ang or MARV/MtE-Mus between 2012 and 2017 at the United States Army Medical Research Institute of Infectious Diseases (USAMRIID). Multivariable Cox proportional hazards regression was used to evaluate the association of variant type with time to death, the development of anorexia, rash, viremia, and 10 select clinical laboratory values. A total of 47 cynomolgus monkeys were included, of which 18 were exposed to MARV/Ang in three separate studies and 29 to MARV/MtE-Mus in five studies. Following universally fatal Marburg virus exposure, compared to MARV/MtE-Mus, MARV/Ang was associated with an increased risk of death (HR = 22.10; 95% CI: 7.08, 68.93), rash (HR = 5.87; 95% CI: 2.76, 12.51) and loss of appetite (HR = 35.10; 95% CI: 7.60, 162.18). Our data demonstrate an increased virulence of MARV/Ang compared to MARV/MtE-Mus variant in the 1000 pfu IM cynomolgus macaque model.

Porcine reproductive and respiratory syndrome virus (PRRSV) is a continuous threat to the pork industry as it continues to cause significant economic loss worldwide. Currently, vaccination strategies provide very limited protection against PRRSV transmission. Consequently, there is an urgent need to develop new antiviral strategies. Platycodin D (PD) is one of the major bioactive triterpenoid saponins derived from Platycodon grandiflorum, a traditional Chinese medicine used as an expectorant for pulmonary diseases and a remedy for respiratory disorders. Here, we demonstrate that PD exhibits potent activity against PRRSV infection in Marc-145 cells and primary porcine alveolar macrophages. PD exhibited broad-spectrum inhibitory activities in vitro against high pathogenic type 2 PRRSV GD-HD strain and GD-XH strain as well as classical CH-1a and VR2332 strains. PD at concentrations ranging 1&amp;ndash;4 &amp;mu;M significantly inhibited PRRSV RNA synthesis, viral protein expression and progeny virus production in a dose-dependent manner. EC50 values of PD against four tested PRRSV strains infection in Marc-145 cells ranged from 0.74 to 1.76 &amp;mu;M. Mechanistically, PD inhibited PRRSV replication by directly interacting with virions therefore affecting multiple stages of the virus life cycle, including viral entry and progeny virus release. In addition, PD decreased PRRSV- and LPS-induced cytokine (IFN-&amp;alpha;, IFN-&amp;beta;, IL-1&amp;alpha;, IL-6, IL-8 and TNF-&amp;alpha;) production in PAMs. Altogether, our findings suggested that PD is a potent inhibitor of PPRSV infection in vitro. However, further in vivo studies are necessary to confirm PD as a potential novel and effective PPRSV inhibitor in swine.

]]>Platycodin D Suppresses Type 2 Porcine Reproductive and Respiratory Syndrome Virus In Primary and Established Cell LinesMingxin ZhangTaofeng DuFeixiang LongXia YangYankuo SunMubing DuanGuihong ZhangYahong LiuEn-min ZhouWeisan ChenJianxin Chendoi: 10.3390/v10110657Viruses2018-11-21Viruses2018-11-211011Article65710.3390/v10110657http://www.mdpi.com/1999-4915/10/11/657Viruses, Vol. 10, Pages 656: Viral Proteins U41 and U70 of Human Herpesvirus 6A Are Dispensable for Telomere Integrationhttp://www.mdpi.com/1999-4915/10/11/656
Human herpesvirus-6A and -6B (HHV-6A and -6B) are two closely related betaherpesviruses that infect humans. Upon primary infection they establish a life-long infection termed latency, where the virus genome is integrated into the telomeres of latently infected cells. Intriguingly, HHV-6A/B can integrate into germ cells, leading to individuals with inherited chromosomally-integrated HHV-6 (iciHHV-6), who have the HHV-6 genome in every cell. It is known that telomeric repeats flanking the virus genome are essential for integration; however, the protein factors mediating integration remain enigmatic. We have previously shown that the putative viral integrase U94 is not essential for telomere integration; thus, we set out to assess the contribution of potential viral recombination proteins U41 and U70 towards integration. We could show that U70 enhances dsDNA break repair via a homology-directed mechanism using a reporter cell line. We then engineered cells to produce shRNAs targeting both U41 and U70 to inhibit their expression during infection. Using these cells in our HHV-6A in vitro integration assay, we could show that U41/U70 were dispensable for telomere integration. Furthermore, additional inhibition of the cellular recombinase Rad51 suggested that it was also not essential, indicating that other cellular and/or viral factors must mediate telomere integration.Viruses, Vol. 10, Pages 656: Viral Proteins U41 and U70 of Human Herpesvirus 6A Are Dispensable for Telomere Integration

Human herpesvirus-6A and -6B (HHV-6A and -6B) are two closely related betaherpesviruses that infect humans. Upon primary infection they establish a life-long infection termed latency, where the virus genome is integrated into the telomeres of latently infected cells. Intriguingly, HHV-6A/B can integrate into germ cells, leading to individuals with inherited chromosomally-integrated HHV-6 (iciHHV-6), who have the HHV-6 genome in every cell. It is known that telomeric repeats flanking the virus genome are essential for integration; however, the protein factors mediating integration remain enigmatic. We have previously shown that the putative viral integrase U94 is not essential for telomere integration; thus, we set out to assess the contribution of potential viral recombination proteins U41 and U70 towards integration. We could show that U70 enhances dsDNA break repair via a homology-directed mechanism using a reporter cell line. We then engineered cells to produce shRNAs targeting both U41 and U70 to inhibit their expression during infection. Using these cells in our HHV-6A in vitro integration assay, we could show that U41/U70 were dispensable for telomere integration. Furthermore, additional inhibition of the cellular recombinase Rad51 suggested that it was also not essential, indicating that other cellular and/or viral factors must mediate telomere integration.

]]>Viral Proteins U41 and U70 of Human Herpesvirus 6A Are Dispensable for Telomere IntegrationDarren J. WightNina WallaschekAnirban SanyalSandra K. WellerLouis FlamandBenedikt B. Kauferdoi: 10.3390/v10110656Viruses2018-11-21Viruses2018-11-211011Article65610.3390/v10110656http://www.mdpi.com/1999-4915/10/11/656Viruses, Vol. 10, Pages 655: Recombinant Lassa Virus Expressing Green Fluorescent Protein as a Tool for High-Throughput Drug Screens and Neutralizing Antibody Assayshttp://www.mdpi.com/1999-4915/10/11/655
Lassa virus (LASV), a mammarenavirus, infects an estimated 100,000&amp;ndash;300,000 individuals yearly in western Africa and frequently causes lethal disease. Currently, no LASV-specific antivirals or vaccines are commercially available for prevention or treatment of Lassa fever, the disease caused by LASV. The development of medical countermeasure screening platforms is a crucial step to yield licensable products. Using reverse genetics, we generated a recombinant wild-type LASV (rLASV-WT) and a modified version thereof encoding a cleavable green fluorescent protein (GFP) as a reporter for rapid and quantitative detection of infection (rLASV-GFP). Both rLASV-WT and wild-type LASV exhibited similar growth kinetics in cultured cells, whereas growth of rLASV-GFP was slightly impaired. GFP reporter expression by rLASV-GFP remained stable over several serial passages in Vero cells. Using two well-characterized broad-spectrum antivirals known to inhibit LASV infection, favipiravir and ribavirin, we demonstrate that rLASV-GFP is a suitable screening tool for the identification of LASV infection inhibitors. Building on these findings, we established a rLASV-GFP-based high-throughput drug discovery screen and an rLASV-GFP-based antibody neutralization assay. Both platforms, now available as a standard tool at the IRF-Frederick (an international resource), will accelerate anti-LASV medical countermeasure discovery and reduce costs of antiviral screens in maximum containment laboratories.Viruses, Vol. 10, Pages 655: Recombinant Lassa Virus Expressing Green Fluorescent Protein as a Tool for High-Throughput Drug Screens and Neutralizing Antibody Assays

Lassa virus (LASV), a mammarenavirus, infects an estimated 100,000&amp;ndash;300,000 individuals yearly in western Africa and frequently causes lethal disease. Currently, no LASV-specific antivirals or vaccines are commercially available for prevention or treatment of Lassa fever, the disease caused by LASV. The development of medical countermeasure screening platforms is a crucial step to yield licensable products. Using reverse genetics, we generated a recombinant wild-type LASV (rLASV-WT) and a modified version thereof encoding a cleavable green fluorescent protein (GFP) as a reporter for rapid and quantitative detection of infection (rLASV-GFP). Both rLASV-WT and wild-type LASV exhibited similar growth kinetics in cultured cells, whereas growth of rLASV-GFP was slightly impaired. GFP reporter expression by rLASV-GFP remained stable over several serial passages in Vero cells. Using two well-characterized broad-spectrum antivirals known to inhibit LASV infection, favipiravir and ribavirin, we demonstrate that rLASV-GFP is a suitable screening tool for the identification of LASV infection inhibitors. Building on these findings, we established a rLASV-GFP-based high-throughput drug discovery screen and an rLASV-GFP-based antibody neutralization assay. Both platforms, now available as a standard tool at the IRF-Frederick (an international resource), will accelerate anti-LASV medical countermeasure discovery and reduce costs of antiviral screens in maximum containment laboratories.

]]>Recombinant Lassa Virus Expressing Green Fluorescent Protein as a Tool for High-Throughput Drug Screens and Neutralizing Antibody AssaysYíngyún CaìMasaharu IwasakiBrett F. BeitzelShuīqìng YúElena N. PostnikovaBeatrice CubittLisa Evans DeWaldSheli R. RadoshitzkyLaura BollingerPeter B. JahrlingGustavo F. PalaciosJuan C. de la TorreJens H. Kuhndoi: 10.3390/v10110655Viruses2018-11-20Viruses2018-11-201011Article65510.3390/v10110655http://www.mdpi.com/1999-4915/10/11/655Viruses, Vol. 10, Pages 654: Description, Distribution, and Relevance of Viruses of the Forest Pathogen Gremmeniella abietinahttp://www.mdpi.com/1999-4915/10/11/654
The European race of the ascomycetous species Gremmeniella abietina (Lagerberg) Morelet includes causal agents of shoot blight and stem canker of several conifers in Europe and North America, which are known to host a diverse virome. GaRV6 is the latest and sixth mycovirus species reported within G. abietina. Before its description, one victorivirus and one gammapartitivirus species were described in biotype A, two mitoviruses in both biotypes A and B and a betaendornavirus in biotype B. Possible phenotypic changes produced by mycoviruses on G. abietina mycelial growth have been reported in Spanish mitovirus-free and GaRV6-hosting G. abietina isolates, which had higher growth rates at the optimal temperature of 15 &amp;deg;C, but no other major differences have been observed between partitivirus-like dsRNA and dsRNA-free isolates. In this review, we reappraise the diversity of viruses found in G. abietina so far, and their relevance in clarifying the taxonomy of G. abietina. We also provide evidence for the presence of two new viruses belonging to the families Fusariviridae and Endornaviridae in Spanish isolates.Viruses, Vol. 10, Pages 654: Description, Distribution, and Relevance of Viruses of the Forest Pathogen Gremmeniella abietina

The European race of the ascomycetous species Gremmeniella abietina (Lagerberg) Morelet includes causal agents of shoot blight and stem canker of several conifers in Europe and North America, which are known to host a diverse virome. GaRV6 is the latest and sixth mycovirus species reported within G. abietina. Before its description, one victorivirus and one gammapartitivirus species were described in biotype A, two mitoviruses in both biotypes A and B and a betaendornavirus in biotype B. Possible phenotypic changes produced by mycoviruses on G. abietina mycelial growth have been reported in Spanish mitovirus-free and GaRV6-hosting G. abietina isolates, which had higher growth rates at the optimal temperature of 15 &amp;deg;C, but no other major differences have been observed between partitivirus-like dsRNA and dsRNA-free isolates. In this review, we reappraise the diversity of viruses found in G. abietina so far, and their relevance in clarifying the taxonomy of G. abietina. We also provide evidence for the presence of two new viruses belonging to the families Fusariviridae and Endornaviridae in Spanish isolates.

]]>Description, Distribution, and Relevance of Viruses of the Forest Pathogen Gremmeniella abietinaLeticia BotellaJarkko Hantuladoi: 10.3390/v10110654Viruses2018-11-20Viruses2018-11-201011Review65410.3390/v10110654http://www.mdpi.com/1999-4915/10/11/654Viruses, Vol. 10, Pages 653: A PB1-K577E Mutation in H9N2 Influenza Virus Increases Polymerase Activity and Pathogenicity in Micehttp://www.mdpi.com/1999-4915/10/11/653
H9N2 avian influenza viruses are present in poultry worldwide. These viruses are considered to have pandemic potential, because recent isolates can recognize human-type receptor and several sporadic human infections have been reported. In this study, we aimed to identify mutations related to mammalian adaptation of H9N2 influenza virus. We found that mouse-adapted viruses had several mutations in hemagglutinin (HA), PB2, PA, and PB1. Among the detected mutations, PB1-K577E was a novel mutation that had not been previously reported to involve mammalian adaptation. A recombinant H9N2 virus bearing only the PB1-K577E mutation showed enhanced pathogenicity in mice, with increased virus titers in nasal turbinates compared to that in mice infected with the wild-type virus. In addition, the PB1-K577E mutation increased virus polymerase activity in human cell culture at a lower temperature. These data suggest that the PB1-K577E mutation is a novel pathogenicity determinant of H9N2 virus in mice and could be a signature for mammalian adaptation.Viruses, Vol. 10, Pages 653: A PB1-K577E Mutation in H9N2 Influenza Virus Increases Polymerase Activity and Pathogenicity in Mice

H9N2 avian influenza viruses are present in poultry worldwide. These viruses are considered to have pandemic potential, because recent isolates can recognize human-type receptor and several sporadic human infections have been reported. In this study, we aimed to identify mutations related to mammalian adaptation of H9N2 influenza virus. We found that mouse-adapted viruses had several mutations in hemagglutinin (HA), PB2, PA, and PB1. Among the detected mutations, PB1-K577E was a novel mutation that had not been previously reported to involve mammalian adaptation. A recombinant H9N2 virus bearing only the PB1-K577E mutation showed enhanced pathogenicity in mice, with increased virus titers in nasal turbinates compared to that in mice infected with the wild-type virus. In addition, the PB1-K577E mutation increased virus polymerase activity in human cell culture at a lower temperature. These data suggest that the PB1-K577E mutation is a novel pathogenicity determinant of H9N2 virus in mice and could be a signature for mammalian adaptation.

]]>A PB1-K577E Mutation in H9N2 Influenza Virus Increases Polymerase Activity and Pathogenicity in MiceHaruhiko KamikiHiromichi MatsugoTomoya KobayashiHiroho IshidaAkiko Takenaka-UemaShin MurakamiTaisuke Horimotodoi: 10.3390/v10110653Viruses2018-11-19Viruses2018-11-191011Article65310.3390/v10110653http://www.mdpi.com/1999-4915/10/11/653Viruses, Vol. 10, Pages 652: Pathobiological and Genomic Characterization of a Cold-Adapted Infectious Bronchitis Virus (BP-caKII)http://www.mdpi.com/1999-4915/10/11/652
We established a cold-adapted infectious bronchitis virus (BP-caKII) by passaging a field virus through specific pathogen-free embryonated eggs 20 times at 32 &amp;deg;C. We characterized its growth kinetics and pathogenicity in embryonated eggs, and its tropism and persistence in different tissues from chickens; then, we evaluated pathogenicity by using a new premature reproductive tract pathogenicity model. Furthermore, we determined the complete genomic sequence of BP-caKII to understand the genetic changes related to cold adaptation. According to our results, BP-caKII clustered with the KII genotype viruses K2 and KM91, and showed less pathogenicity than K2, a live attenuated vaccine strain. BP-caKII showed delayed viremia, resulting in its delayed dissemination to the kidneys and cecal tonsils compared to K2 and KM91, the latter of which is a pathogenic field strain. A comparative genomics study revealed similar nucleotide sequences between BP-caKII, K2 and KM91 but clearly showed different mutations among them. BP-caKII shared several mutations with K2 (nsp13, 14, 15 and 16) following embryo adaptation but acquired multiple additional mutations in nonstructural proteins (nsp3, 4 and 12), spike proteins and nucleocapsid proteins following cold adaptation. Thus, the establishment of BP-caKII and the identified mutations in this study may provide insight into the genetic background of embryo and cold adaptations, and the attenuation of coronaviruses.Viruses, Vol. 10, Pages 652: Pathobiological and Genomic Characterization of a Cold-Adapted Infectious Bronchitis Virus (BP-caKII)

We established a cold-adapted infectious bronchitis virus (BP-caKII) by passaging a field virus through specific pathogen-free embryonated eggs 20 times at 32 &amp;deg;C. We characterized its growth kinetics and pathogenicity in embryonated eggs, and its tropism and persistence in different tissues from chickens; then, we evaluated pathogenicity by using a new premature reproductive tract pathogenicity model. Furthermore, we determined the complete genomic sequence of BP-caKII to understand the genetic changes related to cold adaptation. According to our results, BP-caKII clustered with the KII genotype viruses K2 and KM91, and showed less pathogenicity than K2, a live attenuated vaccine strain. BP-caKII showed delayed viremia, resulting in its delayed dissemination to the kidneys and cecal tonsils compared to K2 and KM91, the latter of which is a pathogenic field strain. A comparative genomics study revealed similar nucleotide sequences between BP-caKII, K2 and KM91 but clearly showed different mutations among them. BP-caKII shared several mutations with K2 (nsp13, 14, 15 and 16) following embryo adaptation but acquired multiple additional mutations in nonstructural proteins (nsp3, 4 and 12), spike proteins and nucleocapsid proteins following cold adaptation. Thus, the establishment of BP-caKII and the identified mutations in this study may provide insight into the genetic background of embryo and cold adaptations, and the attenuation of coronaviruses.

]]>Pathobiological and Genomic Characterization of a Cold-Adapted Infectious Bronchitis Virus (BP-caKII)Seung-Min HongSe-Hee AnChung-Young LeeChang-Seon SongKang-Seuk ChoiJae-Hong KimHyuk-Joon Kwondoi: 10.3390/v10110652Viruses2018-11-19Viruses2018-11-191011Article65210.3390/v10110652http://www.mdpi.com/1999-4915/10/11/652Viruses, Vol. 10, Pages 651: Pathogenesis of Rift Valley Fever Virus Aerosol Infection in STAT2 Knockout Hamstershttp://www.mdpi.com/1999-4915/10/11/651
Rift Valley fever virus (RVFV) is an emerging pathogen capable of causing severe disease in livestock and humans and can be transmitted by multiple routes including aerosol exposure. Several animal models have been developed to gain insight into the pathogenesis associated with aerosolized RVFV infection, but work with these models is restricted to high containment biosafety level (BSL) laboratories limiting their use for antiviral and vaccine development studies. Here, we report on a new RVFV inhalation infection model in STAT2 KO hamsters exposed to aerosolized MP-12 vaccine virus by nose-only inhalation that enables a more accurate delivery and measurement of exposure dose. RVFV was detected in hepatic and other tissues 4&amp;ndash;5 days after challenge, consistent with virus-induced lesions in the liver, spleen and lung. Furthermore, assessment of blood chemistry and hematological parameters revealed alterations in several liver disease markers and white blood cell parameters. Our results indicate that STAT2 KO hamsters develop a disease course that shares features of disease observed in human cases and in other animal models of RVFV aerosol exposure, supporting the use of this BSL-2 infection model for countermeasure development efforts.Viruses, Vol. 10, Pages 651: Pathogenesis of Rift Valley Fever Virus Aerosol Infection in STAT2 Knockout Hamsters

Rift Valley fever virus (RVFV) is an emerging pathogen capable of causing severe disease in livestock and humans and can be transmitted by multiple routes including aerosol exposure. Several animal models have been developed to gain insight into the pathogenesis associated with aerosolized RVFV infection, but work with these models is restricted to high containment biosafety level (BSL) laboratories limiting their use for antiviral and vaccine development studies. Here, we report on a new RVFV inhalation infection model in STAT2 KO hamsters exposed to aerosolized MP-12 vaccine virus by nose-only inhalation that enables a more accurate delivery and measurement of exposure dose. RVFV was detected in hepatic and other tissues 4&amp;ndash;5 days after challenge, consistent with virus-induced lesions in the liver, spleen and lung. Furthermore, assessment of blood chemistry and hematological parameters revealed alterations in several liver disease markers and white blood cell parameters. Our results indicate that STAT2 KO hamsters develop a disease course that shares features of disease observed in human cases and in other animal models of RVFV aerosol exposure, supporting the use of this BSL-2 infection model for countermeasure development efforts.

]]>Pathogenesis of Rift Valley Fever Virus Aerosol Infection in STAT2 Knockout HamstersBrady T. HickersonJonna B. WestoverArnaud J. Van WettereJohanna D. RigasJinxin MiaoBettina L. ConradNeil E. MotterZhongde WangBrian B. Gowendoi: 10.3390/v10110651Viruses2018-11-19Viruses2018-11-191011Article65110.3390/v10110651http://www.mdpi.com/1999-4915/10/11/651Viruses, Vol. 10, Pages 650: Host Lipid Rafts Play a Major Role in Binding and Endocytosis of Influenza A Virushttp://www.mdpi.com/1999-4915/10/11/650
Influenza still remains one of the most challenging diseases, posing a significant threat to public health. Host lipid rafts play a critical role in influenza A virus (IAV) assembly and budding, however, their role in polyvalent IAV host binding and endocytosis had remained elusive until now. In the present study, we observed co-localization of IAV with a lipid raft marker ganglioside, GM1, on the host surface. Further, we isolated the lipid raft micro-domains from IAV infected cells and detected IAV protein in the raft fraction. Finally, raft disruption using Methyl-&amp;beta;-Cyclodextrin revealed significant reduction in IAV host binding, suggesting utilization of host rafts for polyvalent binding on the host cell surface. In addition to this, cyclodextrin mediated inhibition of raft-dependent endocytosis showed significantly reduced IAV internalization. Interestingly, exposure of cells to cyclodextrin two hours post-IAV binding showed no such reduction in IAV entry, indicating use of raft-dependent endocytosis for host entry. In summary, this study demonstrates that host lipid rafts are selected by IAV as a host attachment factors for multivalent binding, and IAV utilizes these micro-domains to exploit raft-dependent endocytosis for host internalization, a virus entry route previously unknown for IAV.Viruses, Vol. 10, Pages 650: Host Lipid Rafts Play a Major Role in Binding and Endocytosis of Influenza A Virus

Influenza still remains one of the most challenging diseases, posing a significant threat to public health. Host lipid rafts play a critical role in influenza A virus (IAV) assembly and budding, however, their role in polyvalent IAV host binding and endocytosis had remained elusive until now. In the present study, we observed co-localization of IAV with a lipid raft marker ganglioside, GM1, on the host surface. Further, we isolated the lipid raft micro-domains from IAV infected cells and detected IAV protein in the raft fraction. Finally, raft disruption using Methyl-&amp;beta;-Cyclodextrin revealed significant reduction in IAV host binding, suggesting utilization of host rafts for polyvalent binding on the host cell surface. In addition to this, cyclodextrin mediated inhibition of raft-dependent endocytosis showed significantly reduced IAV internalization. Interestingly, exposure of cells to cyclodextrin two hours post-IAV binding showed no such reduction in IAV entry, indicating use of raft-dependent endocytosis for host entry. In summary, this study demonstrates that host lipid rafts are selected by IAV as a host attachment factors for multivalent binding, and IAV utilizes these micro-domains to exploit raft-dependent endocytosis for host internalization, a virus entry route previously unknown for IAV.

]]>Host Lipid Rafts Play a Major Role in Binding and Endocytosis of Influenza A VirusDileep Kumar VermaDinesh GuptaSunil Kumar Laldoi: 10.3390/v10110650Viruses2018-11-18Viruses2018-11-181011Article65010.3390/v10110650http://www.mdpi.com/1999-4915/10/11/650Viruses, Vol. 10, Pages 649: Characterizing the Different Effects of Zika Virus Infection in Placenta and Microglia Cellshttp://www.mdpi.com/1999-4915/10/11/649
Zika virus (ZIKV) is a neuropathic virus that causes serious neurological abnormalities such as Guillain-Barre syndrome in adults and congenital Zika syndrome (CZS) in fetuses, which makes it an important concern for global human health. A catalogue of cells that support ZIKV replication, pathogenesis, and/or the persistence of the virus still remains unknown. Here, we studied the behavior of the virus in human placenta (JEG-3) and human microglia (HMC3) cell lines in order to better understand how different host tissues respond during infection. We quantified the host transcriptional response to ZIKV infection in both types of cells at 24 and 72 h post-infection. A panel of 84 genes that are involved in the innate or adaptive immune responses was used to quantify differential expression in both cell lines. HMC3 cells showed a unique set of significant differentially expressed genes (DEGs) compared with JEG-3 cells at both time points. Subsequent analysis of these data using modern pathway analysis methods revealed that the TLR7/8 pathway was strongly inhibited in HMC3 cells, while it was activated in JEG-3 cells during virus infection. The disruption of these pathways was subsequently confirmed with specific small interfering RNA (siRNA) experiments that characterize their role in the viral life cycle, and may partially explain why ZIKV infection in placental tissue contributes to extreme neurological problems in a developing fetus.Viruses, Vol. 10, Pages 649: Characterizing the Different Effects of Zika Virus Infection in Placenta and Microglia Cells

Zika virus (ZIKV) is a neuropathic virus that causes serious neurological abnormalities such as Guillain-Barre syndrome in adults and congenital Zika syndrome (CZS) in fetuses, which makes it an important concern for global human health. A catalogue of cells that support ZIKV replication, pathogenesis, and/or the persistence of the virus still remains unknown. Here, we studied the behavior of the virus in human placenta (JEG-3) and human microglia (HMC3) cell lines in order to better understand how different host tissues respond during infection. We quantified the host transcriptional response to ZIKV infection in both types of cells at 24 and 72 h post-infection. A panel of 84 genes that are involved in the innate or adaptive immune responses was used to quantify differential expression in both cell lines. HMC3 cells showed a unique set of significant differentially expressed genes (DEGs) compared with JEG-3 cells at both time points. Subsequent analysis of these data using modern pathway analysis methods revealed that the TLR7/8 pathway was strongly inhibited in HMC3 cells, while it was activated in JEG-3 cells during virus infection. The disruption of these pathways was subsequently confirmed with specific small interfering RNA (siRNA) experiments that characterize their role in the viral life cycle, and may partially explain why ZIKV infection in placental tissue contributes to extreme neurological problems in a developing fetus.

]]>Characterizing the Different Effects of Zika Virus Infection in Placenta and Microglia CellsMaria del Pilar Martinez ViedmaBrett E. Pickettdoi: 10.3390/v10110649Viruses2018-11-18Viruses2018-11-181011Article64910.3390/v10110649http://www.mdpi.com/1999-4915/10/11/649Viruses, Vol. 10, Pages 648: Willingness to Participate and Associated Factors in a Zika Vaccine Trial in Indonesia: A Cross-Sectional Studyhttp://www.mdpi.com/1999-4915/10/11/648
One of the crucial steps during trials for Zika and other vaccines is to recruit participants and to understand how participants&amp;rsquo; attitudes and sociodemographic characteristics affect willingness to participate (WTP). This study was conducted to assess WTP, its explanatory variables, and the impact of financial compensation on WTP in Indonesia. A health facility-based cross-sectional study was conducted in eleven regencies in the Aceh and West Sumatra provinces of Indonesia. Participants were recruited via a convenience sampling method and were interviewed. The associations between explanatory variables and WTP were assessed using a two-step logistic regression analysis. A total of 1,102 parents were approached, and of these 956 (86.8%) completed the interview and were included in analysis. Of those, 144 (15.1%) were willing to participate in a Zika vaccine trial without a financial compensation. In the multivariate analysis, WTP was tied to an age of more than 50 years old, compared to 20&amp;ndash;29 years (odds ratio (OR): 5.0; 95% confidence interval (CI): 2.37&amp;ndash;10.53), to being female (OR: 2.20; 95% CI: 1.11&amp;ndash;4.37), and to having heard about Zika (OR: 2.41; 95% CI: 1.59&amp;ndash;3.65). Participants&amp;rsquo; WTP increased gradually with higher financial compensation. The rate of WTP increased to 62.3% at the highest offer (US$ 350.4), and those who were still unwilling to participate (37.7%) had a poorer attitude towards childhood vaccination. This study highlights that pre-existing knowledge about Zika and attitudes towards childhood vaccination are important in determining community members being willing to participate in a vaccine trial. Financial incentives are still an important factor to enhance participant recruitment during a vaccine trial.Viruses, Vol. 10, Pages 648: Willingness to Participate and Associated Factors in a Zika Vaccine Trial in Indonesia: A Cross-Sectional Study

One of the crucial steps during trials for Zika and other vaccines is to recruit participants and to understand how participants&amp;rsquo; attitudes and sociodemographic characteristics affect willingness to participate (WTP). This study was conducted to assess WTP, its explanatory variables, and the impact of financial compensation on WTP in Indonesia. A health facility-based cross-sectional study was conducted in eleven regencies in the Aceh and West Sumatra provinces of Indonesia. Participants were recruited via a convenience sampling method and were interviewed. The associations between explanatory variables and WTP were assessed using a two-step logistic regression analysis. A total of 1,102 parents were approached, and of these 956 (86.8%) completed the interview and were included in analysis. Of those, 144 (15.1%) were willing to participate in a Zika vaccine trial without a financial compensation. In the multivariate analysis, WTP was tied to an age of more than 50 years old, compared to 20&amp;ndash;29 years (odds ratio (OR): 5.0; 95% confidence interval (CI): 2.37&amp;ndash;10.53), to being female (OR: 2.20; 95% CI: 1.11&amp;ndash;4.37), and to having heard about Zika (OR: 2.41; 95% CI: 1.59&amp;ndash;3.65). Participants&amp;rsquo; WTP increased gradually with higher financial compensation. The rate of WTP increased to 62.3% at the highest offer (US$ 350.4), and those who were still unwilling to participate (37.7%) had a poorer attitude towards childhood vaccination. This study highlights that pre-existing knowledge about Zika and attitudes towards childhood vaccination are important in determining community members being willing to participate in a vaccine trial. Financial incentives are still an important factor to enhance participant recruitment during a vaccine trial.

Tospovirus is a tripartite negative stranded RNA virus and is considered as one of the most devastating plant viruses. Successful virus infection in plant requires many host factors. To date, very few host factors have been identified as important in Tospovirus infection in plants. We reported earlier that NSm protein encoded by Tomato spotted wilt virus (TSWV), a type species of the genus Orthotospovirus, plays critical roles in viral cell-to-cell and long-distance movement. In this study, we determined that molecular co-chaperone NbSGT1 interacted with TSWV NSm in Nicotiana benthamiana. TSWV infection significantly upregulated the expression of NbSGT1 gene and transient overexpression of NbSGT1 in N. benthamiana leaves accelerated TSWV infection. In contrast, silencing the NbSGT1 gene expression using a virus-induced gene silencing (VIGS) approach strongly inhibited TSWV NSm cell-to-cell movement, as well as TSWV local and systemic infection in N. benthamiana plants. Furthermore, NbSGT1 was found to regulate the infection of both American and Euro/Asia type tospoviruses in N. benthamiana plant. Collectively, our findings presented in this paper and the results published previously indicated that molecular co-chaperone NbSGT1 plays important roles in modulating both positive stranded and tripartite negative stranded RNA virus infection in plants.

]]>Molecular Co-Chaperone SGT1 Is Critical for Cell-to-Cell Movement and Systemic Infection of Tomato Spotted Wilt Virus in Nicotiana benthamianaXin QianQing XiangTongqing YangHongyu MaXin Shun DingXiaorong Taodoi: 10.3390/v10110647Viruses2018-11-17Viruses2018-11-171011Article64710.3390/v10110647http://www.mdpi.com/1999-4915/10/11/647Viruses, Vol. 10, Pages 646: Human Fetal Astrocytes Infected with Zika Virus Exhibit Delayed Apoptosis and Resistance to Interferon: Implications for Persistencehttp://www.mdpi.com/1999-4915/10/11/646
Zika virus (ZIKV) infection and persistence during pregnancy can lead to microcephaly and other fetal neurological disorders collectively known as Congenital Zika Syndrome. The immunological and virological events that contribute to the establishment of persistent ZIKV infection in humans are unclear though. Here we show that human fetal astrocytes (HFAs), the most abundant cell type in the central nervous system, become persistently infected with ZIKV resulting in continuous viral shedding for at least one month; a process that is facilitated by TIM/TAM receptors. HFAs are relatively resistant to ZIKV-induced apoptosis, a factor that may be important for chronic infection of these cells. Once infection was established, interferon treatment did not reduce virus replication. Moreover, the fact that the innate immune system was highly activated in persistently infected HFAs indicates that the virus can thrive in the presence of a sustained antiviral response. RNAseq analyses of persistently infected cells revealed that ZIKV alters host gene expression in a manner that could affect developmental processes. Conversely, data from sequencing of ZIKV genomes in persistently infected HFAs suggest that adaptive mutations were not required for establishing chronic infection. Based on these results, we postulate that HFAs are reservoirs for ZIKV in the fetal brain and that moderate apoptosis combined with inefficient antiviral response from these cells may contribute to the establishment of chronic brain infection associated with the ZIKV neurodevelopmental abnormalities.Viruses, Vol. 10, Pages 646: Human Fetal Astrocytes Infected with Zika Virus Exhibit Delayed Apoptosis and Resistance to Interferon: Implications for Persistence

Zika virus (ZIKV) infection and persistence during pregnancy can lead to microcephaly and other fetal neurological disorders collectively known as Congenital Zika Syndrome. The immunological and virological events that contribute to the establishment of persistent ZIKV infection in humans are unclear though. Here we show that human fetal astrocytes (HFAs), the most abundant cell type in the central nervous system, become persistently infected with ZIKV resulting in continuous viral shedding for at least one month; a process that is facilitated by TIM/TAM receptors. HFAs are relatively resistant to ZIKV-induced apoptosis, a factor that may be important for chronic infection of these cells. Once infection was established, interferon treatment did not reduce virus replication. Moreover, the fact that the innate immune system was highly activated in persistently infected HFAs indicates that the virus can thrive in the presence of a sustained antiviral response. RNAseq analyses of persistently infected cells revealed that ZIKV alters host gene expression in a manner that could affect developmental processes. Conversely, data from sequencing of ZIKV genomes in persistently infected HFAs suggest that adaptive mutations were not required for establishing chronic infection. Based on these results, we postulate that HFAs are reservoirs for ZIKV in the fetal brain and that moderate apoptosis combined with inefficient antiviral response from these cells may contribute to the establishment of chronic brain infection associated with the ZIKV neurodevelopmental abnormalities.

]]>Human Fetal Astrocytes Infected with Zika Virus Exhibit Delayed Apoptosis and Resistance to Interferon: Implications for PersistenceDaniel LimontaJuan JovelAnil KumarAdriana M. AiroShangmei HouLeina SaitoWilliam BrantonGane Ka-Shu WongAndrew MasonChristopher PowerTom C. Hobmandoi: 10.3390/v10110646Viruses2018-11-17Viruses2018-11-171011Article64610.3390/v10110646http://www.mdpi.com/1999-4915/10/11/646Viruses, Vol. 10, Pages 645: HCV-Specific T Cell Responses During and After Chronic HCV Infectionhttp://www.mdpi.com/1999-4915/10/11/645
Hepatitis C virus (HCV)-specific T cell responses are closely linked to the clinical course of infection. While T cell responses in self-limiting infection are typically broad and multi-specific, they display several distinct features of functional impairment in the chronic phase. Moreover, HCV readily adapts to immune pressure by developing escape mutations within epitopes targeted by T cells. Much of our current knowledge on HCV-specific T cell responses has been gathered under the assumption that this might eventually pave the way for a therapeutic vaccine. However, with the development of highly efficient direct acting antivirals (DAAs), there is less interest in the development of a therapeutic vaccine for HCV and the scope of T cell research has shifted. Indeed, the possibility to rapidly eradicate an antigen that has persisted over years or decades, and has led to T cell exhaustion and dysfunction, provides the unique opportunity to study potential T cell recovery after antigen cessation in a human in vivo setting. Findings from such studies not only improve our basic understanding of T cell immunity but may also advance immunotherapeutic approaches in cancer or chronic hepatitis B and D infection. Moreover, in order to edge closer to the WHO goal of HCV elimination by 2030, a prophylactic vaccine is clearly required. Thus, in this review, we will summarize our current knowledge on HCV-specific T cell responses and also provide an outlook on the open questions that require answers in this field.Viruses, Vol. 10, Pages 645: HCV-Specific T Cell Responses During and After Chronic HCV Infection

Hepatitis C virus (HCV)-specific T cell responses are closely linked to the clinical course of infection. While T cell responses in self-limiting infection are typically broad and multi-specific, they display several distinct features of functional impairment in the chronic phase. Moreover, HCV readily adapts to immune pressure by developing escape mutations within epitopes targeted by T cells. Much of our current knowledge on HCV-specific T cell responses has been gathered under the assumption that this might eventually pave the way for a therapeutic vaccine. However, with the development of highly efficient direct acting antivirals (DAAs), there is less interest in the development of a therapeutic vaccine for HCV and the scope of T cell research has shifted. Indeed, the possibility to rapidly eradicate an antigen that has persisted over years or decades, and has led to T cell exhaustion and dysfunction, provides the unique opportunity to study potential T cell recovery after antigen cessation in a human in vivo setting. Findings from such studies not only improve our basic understanding of T cell immunity but may also advance immunotherapeutic approaches in cancer or chronic hepatitis B and D infection. Moreover, in order to edge closer to the WHO goal of HCV elimination by 2030, a prophylactic vaccine is clearly required. Thus, in this review, we will summarize our current knowledge on HCV-specific T cell responses and also provide an outlook on the open questions that require answers in this field.

]]>HCV-Specific T Cell Responses During and After Chronic HCV InfectionHendrik LuxenburgerChristoph Neumann-HaefelinRobert ThimmeTobias Boettlerdoi: 10.3390/v10110645Viruses2018-11-17Viruses2018-11-171011Review64510.3390/v10110645http://www.mdpi.com/1999-4915/10/11/645Viruses, Vol. 10, Pages 644: Site-Specific N-Glycosylation on the AAV8 Capsid Proteinhttp://www.mdpi.com/1999-4915/10/11/644
Adeno associated virus (AAV) is a versatile gene delivery tool, which has been approved as a human gene therapy vector for combating genetic diseases. AAV capsid proteins are the major components that determine the tissue specificity, immunogenicity and in vivo transduction performance of the vector. In this study, the AAV8 capsid glycosylation profile was systemically analyzed by peptide mass fingerprinting utilizing high-resolution mass spectrometry to determine the presence of capsid glycosylation. We identified N-glycosylation on the amino acid N499 of the capsid protein. We characterized the overall sugar profile for vector produced in 293 cells. Multiple N-glycosylated host-cell proteins (HCPs) copurified with AAV8 vectors and were identified by analyzing LC-MS data utilizing a human database and proteome discoverer search engine. The N-glycosylation analysis by MALDI-TOF MS, highlighted the probability of AAV8 interaction with terminal galactosylated N-glycans within the HCPs.Viruses, Vol. 10, Pages 644: Site-Specific N-Glycosylation on the AAV8 Capsid Protein

Adeno associated virus (AAV) is a versatile gene delivery tool, which has been approved as a human gene therapy vector for combating genetic diseases. AAV capsid proteins are the major components that determine the tissue specificity, immunogenicity and in vivo transduction performance of the vector. In this study, the AAV8 capsid glycosylation profile was systemically analyzed by peptide mass fingerprinting utilizing high-resolution mass spectrometry to determine the presence of capsid glycosylation. We identified N-glycosylation on the amino acid N499 of the capsid protein. We characterized the overall sugar profile for vector produced in 293 cells. Multiple N-glycosylated host-cell proteins (HCPs) copurified with AAV8 vectors and were identified by analyzing LC-MS data utilizing a human database and proteome discoverer search engine. The N-glycosylation analysis by MALDI-TOF MS, highlighted the probability of AAV8 interaction with terminal galactosylated N-glycans within the HCPs.

]]>Site-Specific N-Glycosylation on the AAV8 Capsid ProteinArya AloorJunping ZhangEbtesam A. GashashAishwarya ParameswaranMatthew ChrzanowskiCheng MaYong DiaoPeng George WangWeidong Xiaodoi: 10.3390/v10110644Viruses2018-11-17Viruses2018-11-171011Article64410.3390/v10110644http://www.mdpi.com/1999-4915/10/11/644Viruses, Vol. 10, Pages 643: Humanized Mouse Models for the Study of Infection and Pathogenesis of Human Viruseshttp://www.mdpi.com/1999-4915/10/11/643
The evolution of infectious pathogens in humans proved to be a global health problem. Technological advancements over the last 50 years have allowed better means of identifying novel therapeutics to either prevent or combat these infectious diseases. The development of humanized mouse models offers a preclinical in vivo platform for further characterization of human viral infections and human immune responses triggered by these virus particles. Multiple strains of immunocompromised mice reconstituted with a human immune system and/or human hepatocytes are susceptible to infectious pathogens as evidenced by establishment of full viral life cycles in hope of investigating viral&amp;ndash;host interactions observed in patients and discovering potential immunotherapies. This review highlights recent progress in utilizing humanized mice to decipher human specific immune responses against viral tropism.Viruses, Vol. 10, Pages 643: Humanized Mouse Models for the Study of Infection and Pathogenesis of Human Viruses

The evolution of infectious pathogens in humans proved to be a global health problem. Technological advancements over the last 50 years have allowed better means of identifying novel therapeutics to either prevent or combat these infectious diseases. The development of humanized mouse models offers a preclinical in vivo platform for further characterization of human viral infections and human immune responses triggered by these virus particles. Multiple strains of immunocompromised mice reconstituted with a human immune system and/or human hepatocytes are susceptible to infectious pathogens as evidenced by establishment of full viral life cycles in hope of investigating viral&amp;ndash;host interactions observed in patients and discovering potential immunotherapies. This review highlights recent progress in utilizing humanized mice to decipher human specific immune responses against viral tropism.

]]>Humanized Mouse Models for the Study of Infection and Pathogenesis of Human VirusesFritz LaiQingfeng Chendoi: 10.3390/v10110643Viruses2018-11-17Viruses2018-11-171011Review64310.3390/v10110643http://www.mdpi.com/1999-4915/10/11/643Viruses, Vol. 10, Pages 642: Intramuscular Exposure of Macaca fascicularis to Low Doses of Low Passage- or Cell Culture-Adapted Sudan Virus or Ebola Virushttp://www.mdpi.com/1999-4915/10/11/642
The filoviruses Ebola virus (EBOV) and Sudan virus (SUDV) can cause severe diseases, and there are currently no licensed countermeasures available for use against them. Transmission occurs frequently via contact with bodily fluids from infected individuals. However, it can be difficult to determine when or how someone became infected, or the quantity of infectious virus to which they were exposed. Evidence suggests the infectious dose is low, but the majority of published studies use high exposure doses. This study characterized the outcome of exposure to a low dose of EBOV or SUDV, using a Macaca fascicularis model. Further, because the effect of virus passage in cell culture may be more pronounced when lower exposure doses are used, viruses that possessed either the characteristics of wild type viruses (possessing predominantly 7-uridine (7U) genotype and a high particle-to-plaque forming unit (PFU) ratio) or cell culture-passaged viruses (predominantly 8-uridine (8U) genotype, a lower particle-to-PFU ratio) were used. The time to death after a low dose exposure was delayed in comparison to higher exposure doses. These data demonstrated that an extremely low dose of EBOV or SUDV is sufficient to cause lethal disease. A low dose exposure model can help inform studies on pathogenesis, transmission, and optimization of prevention strategies.Viruses, Vol. 10, Pages 642: Intramuscular Exposure of Macaca fascicularis to Low Doses of Low Passage- or Cell Culture-Adapted Sudan Virus or Ebola Virus

The filoviruses Ebola virus (EBOV) and Sudan virus (SUDV) can cause severe diseases, and there are currently no licensed countermeasures available for use against them. Transmission occurs frequently via contact with bodily fluids from infected individuals. However, it can be difficult to determine when or how someone became infected, or the quantity of infectious virus to which they were exposed. Evidence suggests the infectious dose is low, but the majority of published studies use high exposure doses. This study characterized the outcome of exposure to a low dose of EBOV or SUDV, using a Macaca fascicularis model. Further, because the effect of virus passage in cell culture may be more pronounced when lower exposure doses are used, viruses that possessed either the characteristics of wild type viruses (possessing predominantly 7-uridine (7U) genotype and a high particle-to-plaque forming unit (PFU) ratio) or cell culture-passaged viruses (predominantly 8-uridine (8U) genotype, a lower particle-to-PFU ratio) were used. The time to death after a low dose exposure was delayed in comparison to higher exposure doses. These data demonstrated that an extremely low dose of EBOV or SUDV is sufficient to cause lethal disease. A low dose exposure model can help inform studies on pathogenesis, transmission, and optimization of prevention strategies.

]]>Intramuscular Exposure of Macaca fascicularis to Low Doses of Low Passage- or Cell Culture-Adapted Sudan Virus or Ebola VirusKendra J. AlfsonLaura E. AvenaMichael W. BeadlesGabriella WorwaMelanie AmenJean L. PattersonRicardo CarrionAnthony Griffithsdoi: 10.3390/v10110642Viruses2018-11-16Viruses2018-11-161011Article64210.3390/v10110642http://www.mdpi.com/1999-4915/10/11/642Viruses, Vol. 10, Pages 641: Insight into Influenza: A Virus Cap-Snatchinghttp://www.mdpi.com/1999-4915/10/11/641
The influenza A virus (IAV) genome consists of eight single-stranded RNA segments. Each segment is associated with a protein complex, with the 3&amp;prime; and 5&amp;prime; ends bound to the RNA-dependent RNA polymerase (RdRp) and the remainder associated with the viral nucleoprotein. During transcription of viral mRNA, this ribonucleoprotein complex steals short, 5&amp;prime;-capped transcripts produced by the cellular DNA dependent RNA polymerase II (RNAPII) and uses them to prime transcription of viral mRNA. Here, we review the current knowledge on the process of IAV cap-snatching and suggest a requirement for RNAPII promoter-proximal pausing for efficient IAV mRNA transcription.Viruses, Vol. 10, Pages 641: Insight into Influenza: A Virus Cap-Snatching

The influenza A virus (IAV) genome consists of eight single-stranded RNA segments. Each segment is associated with a protein complex, with the 3&amp;prime; and 5&amp;prime; ends bound to the RNA-dependent RNA polymerase (RdRp) and the remainder associated with the viral nucleoprotein. During transcription of viral mRNA, this ribonucleoprotein complex steals short, 5&amp;prime;-capped transcripts produced by the cellular DNA dependent RNA polymerase II (RNAPII) and uses them to prime transcription of viral mRNA. Here, we review the current knowledge on the process of IAV cap-snatching and suggest a requirement for RNAPII promoter-proximal pausing for efficient IAV mRNA transcription.

]]>Insight into Influenza: A Virus Cap-SnatchingCorey De VlugtDorota SikoraMartin Pelchatdoi: 10.3390/v10110641Viruses2018-11-16Viruses2018-11-161011Review64110.3390/v10110641http://www.mdpi.com/1999-4915/10/11/641Viruses, Vol. 10, Pages 640: IP6 Regulation of HIV Capsid Assembly, Stability, and Uncoatinghttp://www.mdpi.com/1999-4915/10/11/640
The mechanisms that drive formation of the HIV capsid, first as an immature particle and then as a mature protein shell, remain incompletely understood. Recent discoveries of positively-charged rings in the immature and mature protein hexamer subunits that comprise them and their binding to the cellular metabolite inositol hexakisphosphate (IP6) have stimulated exciting new hypotheses. In this paper, we discuss how data from multiple structural and biochemical approaches are revealing potential roles for IP6 in the HIV-1 replication cycle from assembly to uncoating.Viruses, Vol. 10, Pages 640: IP6 Regulation of HIV Capsid Assembly, Stability, and Uncoating

The mechanisms that drive formation of the HIV capsid, first as an immature particle and then as a mature protein shell, remain incompletely understood. Recent discoveries of positively-charged rings in the immature and mature protein hexamer subunits that comprise them and their binding to the cellular metabolite inositol hexakisphosphate (IP6) have stimulated exciting new hypotheses. In this paper, we discuss how data from multiple structural and biochemical approaches are revealing potential roles for IP6 in the HIV-1 replication cycle from assembly to uncoating.

]]>IP6 Regulation of HIV Capsid Assembly, Stability, and UncoatingRobert A. DickDonna L. MalleryVolker M. VogtLeo C. Jamesdoi: 10.3390/v10110640Viruses2018-11-15Viruses2018-11-151011Review64010.3390/v10110640http://www.mdpi.com/1999-4915/10/11/640Viruses, Vol. 10, Pages 639: Human Bocavirus Infection Markers in Peripheral Blood and Stool Samples of Children with Acute Gastroenteritishttp://www.mdpi.com/1999-4915/10/11/639
Human bocaviruses (HBoVs) 1&amp;ndash;4 belong to the Parvoviridae family, and they infect the respiratory or gastrointestinal tracts in children. We investigated the prevalence of HBoV1&amp;ndash;4 DNAs in the blood and stool samples, and of HBoV1&amp;ndash;4 IgG and IgM in the plasma samples, of children presenting with acute gastroenteritis (AGE). In addition, we identified HBoV co-infections with the five most frequent gastrointestinal pathogens. A total of 83 paired blood and stool samples were collected from children aged five years or less. Infection markers of HBoV1, 2, or 3 (viral DNA in blood and/or stool and/or antibodies) were detected in 61 out of 83 (73.5%) patients. HBoV1, 2, or 3 DNA as a monoinfection was revealed in 18.1%, 2.4%, and 1.2%, respectively, and 21.7% in total. In 56.1% of the HBoV DNA-positive patients, the presence in stool of another virus&amp;mdash;most frequently norovirus or rotavirus&amp;mdash;was observed. In conclusion, this study, for the first time, illustrates the prevalence and genetic diversity of HBoVs in Latvian children with gastroenteritis, and shows a widespread distribution of these viruses in the community. HBoV1 and 2 are commonly found as single infectious agents in children with AGE, suggesting that the viruses can be as pathogenic by themselves as other enteric agents are.Viruses, Vol. 10, Pages 639: Human Bocavirus Infection Markers in Peripheral Blood and Stool Samples of Children with Acute Gastroenteritis

Human bocaviruses (HBoVs) 1&amp;ndash;4 belong to the Parvoviridae family, and they infect the respiratory or gastrointestinal tracts in children. We investigated the prevalence of HBoV1&amp;ndash;4 DNAs in the blood and stool samples, and of HBoV1&amp;ndash;4 IgG and IgM in the plasma samples, of children presenting with acute gastroenteritis (AGE). In addition, we identified HBoV co-infections with the five most frequent gastrointestinal pathogens. A total of 83 paired blood and stool samples were collected from children aged five years or less. Infection markers of HBoV1, 2, or 3 (viral DNA in blood and/or stool and/or antibodies) were detected in 61 out of 83 (73.5%) patients. HBoV1, 2, or 3 DNA as a monoinfection was revealed in 18.1%, 2.4%, and 1.2%, respectively, and 21.7% in total. In 56.1% of the HBoV DNA-positive patients, the presence in stool of another virus&amp;mdash;most frequently norovirus or rotavirus&amp;mdash;was observed. In conclusion, this study, for the first time, illustrates the prevalence and genetic diversity of HBoVs in Latvian children with gastroenteritis, and shows a widespread distribution of these viruses in the community. HBoV1 and 2 are commonly found as single infectious agents in children with AGE, suggesting that the viruses can be as pathogenic by themselves as other enteric agents are.

]]>Human Bocavirus Infection Markers in Peripheral Blood and Stool Samples of Children with Acute GastroenteritisZaiga Nora-KrukleAnda VilmaneMan XuSanta RasaInga ZiemeleElina SilinaMaria Söderlund-VenermoDace GardovskaModra Murovskadoi: 10.3390/v10110639Viruses2018-11-15Viruses2018-11-151011Article63910.3390/v10110639http://www.mdpi.com/1999-4915/10/11/639Viruses, Vol. 10, Pages 638: Safety Studies of Pneumococcal Endolysins Cpl-1 and Palhttp://www.mdpi.com/1999-4915/10/11/638
Bacteriophage-derived endolysins have gained increasing attention as potent antimicrobial agents and numerous publications document the in vivo efficacy of these enzymes in various rodent models. However, little has been documented about their safety and toxicity profiles. Here, we present preclinical safety and toxicity data for two pneumococcal endolysins, Pal and Cpl-1. Microarray, and gene profiling was performed on human macrophages and pharyngeal cells exposed to 0.5 &amp;micro;M of each endolysin for six hours and no change in gene expression was noted. Likewise, in mice injected with 15 mg/kg of each endolysin, no physical or behavioral changes were noted, pro-inflammatory cytokine levels remained constant, and there were no significant changes in the fecal microbiome. Neither endolysin caused complement activation via the classic pathway, the alternative pathway, or the mannose-binding lectin pathway. In cellular response assays, IgG levels in mice exposed to Pal or Cpl-1 gradually increased for the first 30 days post exposure, but IgE levels never rose above baseline, suggesting that hypersensitivity or allergic reaction is unlikely. Collectively, the safety and toxicity profiles of Pal and Cpl-1 support further preclinical studies.Viruses, Vol. 10, Pages 638: Safety Studies of Pneumococcal Endolysins Cpl-1 and Pal

Bacteriophage-derived endolysins have gained increasing attention as potent antimicrobial agents and numerous publications document the in vivo efficacy of these enzymes in various rodent models. However, little has been documented about their safety and toxicity profiles. Here, we present preclinical safety and toxicity data for two pneumococcal endolysins, Pal and Cpl-1. Microarray, and gene profiling was performed on human macrophages and pharyngeal cells exposed to 0.5 &amp;micro;M of each endolysin for six hours and no change in gene expression was noted. Likewise, in mice injected with 15 mg/kg of each endolysin, no physical or behavioral changes were noted, pro-inflammatory cytokine levels remained constant, and there were no significant changes in the fecal microbiome. Neither endolysin caused complement activation via the classic pathway, the alternative pathway, or the mannose-binding lectin pathway. In cellular response assays, IgG levels in mice exposed to Pal or Cpl-1 gradually increased for the first 30 days post exposure, but IgE levels never rose above baseline, suggesting that hypersensitivity or allergic reaction is unlikely. Collectively, the safety and toxicity profiles of Pal and Cpl-1 support further preclinical studies.

]]>Safety Studies of Pneumococcal Endolysins Cpl-1 and PalMarek HarhalaDaniel C. NelsonPaulina MiernikiewiczRyan D. HeselpothBeata BrzezickaJoanna MajewskaSara B. LindenXiaoran ShangAleksander SzymczakDorota LecionKarolina Marek-BukowiecMarlena KłakBartosz WojciechowiczKarolina LahuttaAndrzej KoniecznyKrystyna Dąbrowskadoi: 10.3390/v10110638Viruses2018-11-15Viruses2018-11-151011Article63810.3390/v10110638http://www.mdpi.com/1999-4915/10/11/638Viruses, Vol. 10, Pages 637: Base-By-Base Version 3: New Comparative Tools for Large Virus Genomeshttp://www.mdpi.com/1999-4915/10/11/637
Base-By-Base is a comprehensive tool for the creation and editing of multiple sequence alignments that is coded in Java and runs on multiple platforms. It can be used with gene and protein sequences as well as with large viral genomes, which themselves can contain gene annotations. This report describes new features added to Base-By-Base over the last 7 years. The two most significant additions are: (1) The recoding and inclusion of &amp;ldquo;consensus-degenerate hybrid oligonucleotide primers&amp;rdquo; (CODEHOP), a popular tool for the design of degenerate primers from a multiple sequence alignment of proteins; and (2) the ability to perform fuzzy searches within the columns of sequence data in multiple sequence alignments to determine the distribution of sequence variants among the sequences. The intuitive interface focuses on the presentation of results in easily understood visualizations and providing the ability to annotate the sequences in a multiple alignment with analytic and user data.Viruses, Vol. 10, Pages 637: Base-By-Base Version 3: New Comparative Tools for Large Virus Genomes

Base-By-Base is a comprehensive tool for the creation and editing of multiple sequence alignments that is coded in Java and runs on multiple platforms. It can be used with gene and protein sequences as well as with large viral genomes, which themselves can contain gene annotations. This report describes new features added to Base-By-Base over the last 7 years. The two most significant additions are: (1) The recoding and inclusion of &amp;ldquo;consensus-degenerate hybrid oligonucleotide primers&amp;rdquo; (CODEHOP), a popular tool for the design of degenerate primers from a multiple sequence alignment of proteins; and (2) the ability to perform fuzzy searches within the columns of sequence data in multiple sequence alignments to determine the distribution of sequence variants among the sequences. The intuitive interface focuses on the presentation of results in easily understood visualizations and providing the ability to annotate the sequences in a multiple alignment with analytic and user data.

]]>Base-By-Base Version 3: New Comparative Tools for Large Virus GenomesShin-Lin TuJeannette P. StaheliColum McClayKathleen McLeodTimothy M. RoseChris Uptondoi: 10.3390/v10110637Viruses2018-11-15Viruses2018-11-151011Article63710.3390/v10110637http://www.mdpi.com/1999-4915/10/11/637Viruses, Vol. 10, Pages 636: Glycans Controlling Virus Infections: Meeting Report on the 1st International Symposium on Glycovirology Schöntal, Germany, 02–04 May 2018http://www.mdpi.com/1999-4915/10/11/636
Glycans are, with nucleic acids, proteins and lipids, one of the four founding structures of cellular life. Due to their non-template synthesis, they are inherently heterogeneous and difficult to study with regards to their structure and function. Since 2016, the research group ViroCarb, funded by the German Research Foundation, has investigated the role of glycans in non-enveloped virus infections with a highly interdisciplinary approach. The core idea was to bring together scientists and students from various disciplines such as structural biology, cell biology, virology and chemistry to advance research by an interdisciplinary means. In 2018, ViroCarb hosted the 1st International Symposium on Glycovirology in Sch&amp;ouml;ntal, Germany, with a similar aim. Scientists from various disciplines gathered to discuss their area of study, present recent findings, establish or strengthen collaborations, and mentor the next generation of glycovirologists through formal presentations and informal discussions. The secluded meeting at the monastery of Sch&amp;ouml;ntal gave ample time for in-depth discussions. On behalf of ViroCarb, this report summarizes the reports and highlights advances in the field.Viruses, Vol. 10, Pages 636: Glycans Controlling Virus Infections: Meeting Report on the 1st International Symposium on Glycovirology Schöntal, Germany, 02–04 May 2018

Glycans are, with nucleic acids, proteins and lipids, one of the four founding structures of cellular life. Due to their non-template synthesis, they are inherently heterogeneous and difficult to study with regards to their structure and function. Since 2016, the research group ViroCarb, funded by the German Research Foundation, has investigated the role of glycans in non-enveloped virus infections with a highly interdisciplinary approach. The core idea was to bring together scientists and students from various disciplines such as structural biology, cell biology, virology and chemistry to advance research by an interdisciplinary means. In 2018, ViroCarb hosted the 1st International Symposium on Glycovirology in Sch&amp;ouml;ntal, Germany, with a similar aim. Scientists from various disciplines gathered to discuss their area of study, present recent findings, establish or strengthen collaborations, and mentor the next generation of glycovirologists through formal presentations and informal discussions. The secluded meeting at the monastery of Sch&amp;ouml;ntal gave ample time for in-depth discussions. On behalf of ViroCarb, this report summarizes the reports and highlights advances in the field.

]]>Glycans Controlling Virus Infections: Meeting Report on the 1st International Symposium on Glycovirology Schöntal, Germany, 02–04 May 2018Thilo StehleThomas PetersLaura HartmannMario Schelhaasdoi: 10.3390/v10110636Viruses2018-11-15Viruses2018-11-151011Meeting Report63610.3390/v10110636http://www.mdpi.com/1999-4915/10/11/636Viruses, Vol. 10, Pages 635: The In Ovo Delivery of CpG Oligonucleotides Protects against Infectious Bronchitis with the Recruitment of Immune Cells into the Respiratory Tract of Chickenshttp://www.mdpi.com/1999-4915/10/11/635
The in ovo delivery of cytosine-guanosine (CpG) oligodeoxynucleotides (ODNs) protects chickens against many bacterial and viral infections, by activating the toll-like receptor (TLR)21 signaling pathway. Although the delivery of CpG ODNs in ovo at embryo day (ED) 18 has been shown to reduce infectious bronchitis virus (IBV) loads in embryonic chicken lungs pre-hatch, whether in ovo delivered CpG ODNs are capable of protecting chickens against a post-hatch challenge is unknown. Thus, our objectives were to determine the protective effect of the in ovo delivery of CpG ODNs at ED 18 against IBV infection encountered post-hatch and, then, to investigate the mechanisms of protection. We found significantly higher survival rates and reduced IBV infection in the chickens following the pre-treatment of the ED 18 eggs with CpG ODNs. At 3 days post infection (dpi), we found an increased recruitment of macrophages, cluster of differentiation (CD)8&amp;alpha;+ and CD4+ T lymphocytes, and an up-regulation of interferon (IFN)-&amp;gamma; mRNA in the respiratory tract of the chickens. Overall, it may be inferred that CpG ODNs, when delivered in ovo, provide protection against IBV infection induced morbidity and mortality with an enhanced immune response.Viruses, Vol. 10, Pages 635: The In Ovo Delivery of CpG Oligonucleotides Protects against Infectious Bronchitis with the Recruitment of Immune Cells into the Respiratory Tract of Chickens

The in ovo delivery of cytosine-guanosine (CpG) oligodeoxynucleotides (ODNs) protects chickens against many bacterial and viral infections, by activating the toll-like receptor (TLR)21 signaling pathway. Although the delivery of CpG ODNs in ovo at embryo day (ED) 18 has been shown to reduce infectious bronchitis virus (IBV) loads in embryonic chicken lungs pre-hatch, whether in ovo delivered CpG ODNs are capable of protecting chickens against a post-hatch challenge is unknown. Thus, our objectives were to determine the protective effect of the in ovo delivery of CpG ODNs at ED 18 against IBV infection encountered post-hatch and, then, to investigate the mechanisms of protection. We found significantly higher survival rates and reduced IBV infection in the chickens following the pre-treatment of the ED 18 eggs with CpG ODNs. At 3 days post infection (dpi), we found an increased recruitment of macrophages, cluster of differentiation (CD)8&amp;alpha;+ and CD4+ T lymphocytes, and an up-regulation of interferon (IFN)-&amp;gamma; mRNA in the respiratory tract of the chickens. Overall, it may be inferred that CpG ODNs, when delivered in ovo, provide protection against IBV infection induced morbidity and mortality with an enhanced immune response.

]]>The In Ovo Delivery of CpG Oligonucleotides Protects against Infectious Bronchitis with the Recruitment of Immune Cells into the Respiratory Tract of ChickensUpasama De Silva SenapathiMohamed Sarjoon Abdul-CaderAruna AmarasingheGuido van MarleMarkus CzubSusantha GomisMohamed Faizal Abdul-Careemdoi: 10.3390/v10110635Viruses2018-11-15Viruses2018-11-151011Communication63510.3390/v10110635http://www.mdpi.com/1999-4915/10/11/635Viruses, Vol. 10, Pages 634: Development and Characterization of Double-Antibody Sandwich ELISA for Detection of Zika Virus Infectionhttp://www.mdpi.com/1999-4915/10/11/634
Zika virus (ZIKV) is an emerging mosquito-transmitted flavivirus that can cause severe disease, including congenital birth defect and Guillain&amp;minus;Barr&amp;eacute; syndrome during pregnancy. Although, several molecular diagnostic methods have been developed to detect the ZIKV, these methods pose challenges as they cannot detect early viral infection. Furthermore, these methods require the extraction of RNA, which is easy to contaminate. Nonstructural protein 1 (NS1) is an important biomarker for early diagnosis of the virus, and the detection methods associated with the NS1 protein have recently been reported. The aim of this study was to develop a rapid and sensitive detection method for the detection of the ZIKV based on the NS1 protein. The sensitivity of this method is 120 ng mL&amp;minus;1 and it detected the ZIKV in the supernatant and lysates of Vero and BHK cells, as well as the sera of tree shrews infected with the ZIKV. Without the isolation of the virus and the extraction of the RNA, our method can be used as a primary screening test as opposed to other diagnosis methods that detect the ZIKV.Viruses, Vol. 10, Pages 634: Development and Characterization of Double-Antibody Sandwich ELISA for Detection of Zika Virus Infection

Zika virus (ZIKV) is an emerging mosquito-transmitted flavivirus that can cause severe disease, including congenital birth defect and Guillain&amp;minus;Barr&amp;eacute; syndrome during pregnancy. Although, several molecular diagnostic methods have been developed to detect the ZIKV, these methods pose challenges as they cannot detect early viral infection. Furthermore, these methods require the extraction of RNA, which is easy to contaminate. Nonstructural protein 1 (NS1) is an important biomarker for early diagnosis of the virus, and the detection methods associated with the NS1 protein have recently been reported. The aim of this study was to develop a rapid and sensitive detection method for the detection of the ZIKV based on the NS1 protein. The sensitivity of this method is 120 ng mL&amp;minus;1 and it detected the ZIKV in the supernatant and lysates of Vero and BHK cells, as well as the sera of tree shrews infected with the ZIKV. Without the isolation of the virus and the extraction of the RNA, our method can be used as a primary screening test as opposed to other diagnosis methods that detect the ZIKV.

]]>Development and Characterization of Double-Antibody Sandwich ELISA for Detection of Zika Virus InfectionLiding ZhangXuewei DuCongjie ChenZhixin ChenLi ZhangQinqin HanXueshan XiaYuzhu SongJinyang Zhangdoi: 10.3390/v10110634Viruses2018-11-15Viruses2018-11-151011Article63410.3390/v10110634http://www.mdpi.com/1999-4915/10/11/634Viruses, Vol. 10, Pages 633: Metagenomic Next-Generation Sequencing Reveals Individual Composition and Dynamics of Anelloviruses during Autologous Stem Cell Transplant Recipient Managementhttp://www.mdpi.com/1999-4915/10/11/633
Over recent years, there has been increasing interest in the use of the anelloviruses, the major component of the human virome, for the prediction of post-transplant complications such as severe infections. Due to an important diversity, the comprehensive characterization of this viral family over time has been poorly studied. To overcome this challenge, we used a metagenomic next-generation sequencing (mNGS) approach with the aim of determining the individual anellovirus profile of autologous stem cell transplant (ASCT) patients. We conducted a prospective pilot study on a homogeneous patient cohort regarding the chemotherapy regimens that included 10 ASCT recipients. A validated viral mNGS workflow was used on 108 plasma samples collected at 11 time points from diagnosis to 90 days post-transplantation. A complex interindividual variability in terms of abundance and composition was noticed. In particular, a strong sex effect was found and confirmed using quantitative PCR targeting torque teno virus, the most abundant anellovirus. Interestingly, an important turnover in the anellovirus composition was observed during the course of the disease revealing a strong intra-individual variability. Although more studies are needed to better understand anellovirus dynamics, these findings are of prime importance for their future use as biomarkers of immune competence.Viruses, Vol. 10, Pages 633: Metagenomic Next-Generation Sequencing Reveals Individual Composition and Dynamics of Anelloviruses during Autologous Stem Cell Transplant Recipient Management

Over recent years, there has been increasing interest in the use of the anelloviruses, the major component of the human virome, for the prediction of post-transplant complications such as severe infections. Due to an important diversity, the comprehensive characterization of this viral family over time has been poorly studied. To overcome this challenge, we used a metagenomic next-generation sequencing (mNGS) approach with the aim of determining the individual anellovirus profile of autologous stem cell transplant (ASCT) patients. We conducted a prospective pilot study on a homogeneous patient cohort regarding the chemotherapy regimens that included 10 ASCT recipients. A validated viral mNGS workflow was used on 108 plasma samples collected at 11 time points from diagnosis to 90 days post-transplantation. A complex interindividual variability in terms of abundance and composition was noticed. In particular, a strong sex effect was found and confirmed using quantitative PCR targeting torque teno virus, the most abundant anellovirus. Interestingly, an important turnover in the anellovirus composition was observed during the course of the disease revealing a strong intra-individual variability. Although more studies are needed to better understand anellovirus dynamics, these findings are of prime importance for their future use as biomarkers of immune competence.

]]>Metagenomic Next-Generation Sequencing Reveals Individual Composition and Dynamics of Anelloviruses during Autologous Stem Cell Transplant Recipient ManagementAntonin BalClémentine SarkozyLaurence JossetValérie CheynetGuy OriolJérémie BeckerGaëlle VilchezPierre SesquesFrançois MalletAlexandre PachotFlorence MorfinBruno LinaGilles SallesFréderic ReynierSophie Trouillet-AssantKaren Brengel-Pescedoi: 10.3390/v10110633Viruses2018-11-14Viruses2018-11-141011Communication63310.3390/v10110633http://www.mdpi.com/1999-4915/10/11/633Viruses, Vol. 10, Pages 630: TIM-1 Promotes Japanese Encephalitis Virus Entry and Infectionhttp://www.mdpi.com/1999-4915/10/11/630
Japanese encephalitis virus (JEV) is a mosquito-borne Flavivirus, the leading cause of viral-induced encephalitis. Several host molecules have been identified as the JEV attachment factor; however, the molecules involved in JEV entry remain poorly understood. In the present study, we demonstrate that TIM-1 is important for efficient infection by JEV. Firstly, three TIM-1 variants (V1, V2, and V3) were cloned from A549 cells, and we revealed that only ectopically TIM-1 V2 expression in 293T cells significantly promotes JEV attachment, entry and infection. Point mutation of phosphatidylserine (Ptdser) binding pocket in the TIM-1 IgV domain dampened JEV entry, indicating that TIM-1-mediated JEV infection is Ptdser-dependent. Furthermore, we found the cytoplasmic domain of TIM-1 is also required for enhancing JEV entry. Additionally, knock down of TIM-1 expression in A549 cells impaired JEV entry and infection, but not attachment, suggesting that additional factors exist in A549 cells that allow the virus to bind. In conclusion, our findings demonstrate that TIM-1 promotes JEV infection as an entry cofactor, and the polymorphism of TIM-1 is associated with JEV susceptibility to host cells.Viruses, Vol. 10, Pages 630: TIM-1 Promotes Japanese Encephalitis Virus Entry and Infection

Japanese encephalitis virus (JEV) is a mosquito-borne Flavivirus, the leading cause of viral-induced encephalitis. Several host molecules have been identified as the JEV attachment factor; however, the molecules involved in JEV entry remain poorly understood. In the present study, we demonstrate that TIM-1 is important for efficient infection by JEV. Firstly, three TIM-1 variants (V1, V2, and V3) were cloned from A549 cells, and we revealed that only ectopically TIM-1 V2 expression in 293T cells significantly promotes JEV attachment, entry and infection. Point mutation of phosphatidylserine (Ptdser) binding pocket in the TIM-1 IgV domain dampened JEV entry, indicating that TIM-1-mediated JEV infection is Ptdser-dependent. Furthermore, we found the cytoplasmic domain of TIM-1 is also required for enhancing JEV entry. Additionally, knock down of TIM-1 expression in A549 cells impaired JEV entry and infection, but not attachment, suggesting that additional factors exist in A549 cells that allow the virus to bind. In conclusion, our findings demonstrate that TIM-1 promotes JEV infection as an entry cofactor, and the polymorphism of TIM-1 is associated with JEV susceptibility to host cells.

]]>TIM-1 Promotes Japanese Encephalitis Virus Entry and InfectionJichen NiuYa JiangHao XuChangjing ZhaoGuodong ZhouPuyan ChenRuibing Caodoi: 10.3390/v10110630Viruses2018-11-14Viruses2018-11-141011Article63010.3390/v10110630http://www.mdpi.com/1999-4915/10/11/630Viruses, Vol. 10, Pages 632: Autographa Californica Multiple Nucleopolyhedrovirus Enters Host Cells via Clathrin-Mediated Endocytosis and Direct Fusion with the Plasma Membranehttp://www.mdpi.com/1999-4915/10/11/632
The cell entry mechanism of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is not fully understood. Previous studies showed that AcMNPV entered host cells primarily through clathrin-mediated endocytosis, and could efficiently infect cells via fusion with the plasma membrane after a low-pH trigger. However, whether AcMNPV enters cells via these two pathways simultaneously, and the exact manner in which AcMNPV particles are internalized into cells remains unclear. In this study, using single-virus tracking, we observed that AcMNPV particles were first captured by pre-existing clathrin-coated pits (CCP), and were then delivered to early endosomes. Population-based analysis of single-virus tracking and quantitative electron microscopy demonstrated that the majority of particles were captured by CCPs and internalized via invagination. In contrast, a minority of virus particles were not delivered to CCPs, and were internalized through direct fusion with the plasma membrane without invagination. Quantitative electron microscopy also showed that, while inhibition of CCP assembly significantly impaired viral internalization, inhibition of endosomal acidification blocked virus particles out of vesicles. Collectively, these findings demonstrated that approximately 90% of AcMNPV particles entered cells through clathrin-mediated endocytosis and 10% entered via direct fusion with the plasma membrane. This study will lead toward a better understanding of AcMNPV infection.Viruses, Vol. 10, Pages 632: Autographa Californica Multiple Nucleopolyhedrovirus Enters Host Cells via Clathrin-Mediated Endocytosis and Direct Fusion with the Plasma Membrane

The cell entry mechanism of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is not fully understood. Previous studies showed that AcMNPV entered host cells primarily through clathrin-mediated endocytosis, and could efficiently infect cells via fusion with the plasma membrane after a low-pH trigger. However, whether AcMNPV enters cells via these two pathways simultaneously, and the exact manner in which AcMNPV particles are internalized into cells remains unclear. In this study, using single-virus tracking, we observed that AcMNPV particles were first captured by pre-existing clathrin-coated pits (CCP), and were then delivered to early endosomes. Population-based analysis of single-virus tracking and quantitative electron microscopy demonstrated that the majority of particles were captured by CCPs and internalized via invagination. In contrast, a minority of virus particles were not delivered to CCPs, and were internalized through direct fusion with the plasma membrane without invagination. Quantitative electron microscopy also showed that, while inhibition of CCP assembly significantly impaired viral internalization, inhibition of endosomal acidification blocked virus particles out of vesicles. Collectively, these findings demonstrated that approximately 90% of AcMNPV particles entered cells through clathrin-mediated endocytosis and 10% entered via direct fusion with the plasma membrane. This study will lead toward a better understanding of AcMNPV infection.

]]>Autographa Californica Multiple Nucleopolyhedrovirus Enters Host Cells via Clathrin-Mediated Endocytosis and Direct Fusion with the Plasma MembraneFujun QinCongrui XuChengfeng LeiJia HuXiulian Sundoi: 10.3390/v10110632Viruses2018-11-14Viruses2018-11-141011Article63210.3390/v10110632http://www.mdpi.com/1999-4915/10/11/632Viruses, Vol. 10, Pages 631: Recent Advances in Zika Virus Vaccineshttp://www.mdpi.com/1999-4915/10/11/631
The recent outbreaks of Zika virus (ZIKV) infections and associated microcephaly in newborns has resulted in an unprecedented effort by researchers to target this virus. Significant advances have been made in developing vaccine candidates, treatment strategies and diagnostic assays in a relatively short period of time. Being a preventable disease, the first line of defense against ZIKV would be to vaccinate the highly susceptible target population, especially pregnant women. Along those lines, several vaccine candidates including purified inactivated virus (PIV), live attenuated virus (LAV), virus like particles (VLP), DNA, modified RNA, viral vectors and subunit vaccines have been in the pipeline with several advancing to clinical trials. As the primary objective of Zika vaccination is the prevention of vertical transmission of the virus to the unborn fetus, the safety and efficacy requirements for this vaccine remain unique when compared to other diseases. This review will discuss these recent advances in the field of Zika vaccine development.Viruses, Vol. 10, Pages 631: Recent Advances in Zika Virus Vaccines

The recent outbreaks of Zika virus (ZIKV) infections and associated microcephaly in newborns has resulted in an unprecedented effort by researchers to target this virus. Significant advances have been made in developing vaccine candidates, treatment strategies and diagnostic assays in a relatively short period of time. Being a preventable disease, the first line of defense against ZIKV would be to vaccinate the highly susceptible target population, especially pregnant women. Along those lines, several vaccine candidates including purified inactivated virus (PIV), live attenuated virus (LAV), virus like particles (VLP), DNA, modified RNA, viral vectors and subunit vaccines have been in the pipeline with several advancing to clinical trials. As the primary objective of Zika vaccination is the prevention of vertical transmission of the virus to the unborn fetus, the safety and efficacy requirements for this vaccine remain unique when compared to other diseases. This review will discuss these recent advances in the field of Zika vaccine development.

]]>Recent Advances in Zika Virus VaccinesHimanshu GargTugba Mehmetoglu-GurbuzAnjali Joshidoi: 10.3390/v10110631Viruses2018-11-14Viruses2018-11-141011Review63110.3390/v10110631http://www.mdpi.com/1999-4915/10/11/631Viruses, Vol. 10, Pages 629: ISG15, a Small Molecule with Huge Implications: Regulation of Mitochondrial Homeostasishttp://www.mdpi.com/1999-4915/10/11/629
Viruses are responsible for the majority of infectious diseases, from the common cold to HIV/AIDS or hemorrhagic fevers, the latter with devastating effects on the human population. Accordingly, the development of efficient antiviral therapies is a major goal and a challenge for the scientific community, as we are still far from understanding the molecular mechanisms that operate after virus infection. Interferon-stimulated gene 15 (ISG15) plays an important antiviral role during viral infection. ISG15 catalyzes a ubiquitin-like post-translational modification termed ISGylation, involving the conjugation of ISG15 molecules to de novo synthesized viral or cellular proteins, which regulates their stability and function. Numerous biomedically relevant viruses are targets of ISG15, as well as proteins involved in antiviral immunity. Beyond their role as cellular powerhouses, mitochondria are multifunctional organelles that act as signaling hubs in antiviral responses. In this review, we give an overview of the biological consequences of ISGylation for virus infection and host defense. We also compare several published proteomic studies to identify and classify potential mitochondrial ISGylation targets. Finally, based on our recent observations, we discuss the essential functions of mitochondria in the antiviral response and examine the role of ISG15 in the regulation of mitochondrial processes, specifically OXPHOS and mitophagy.Viruses, Vol. 10, Pages 629: ISG15, a Small Molecule with Huge Implications: Regulation of Mitochondrial Homeostasis

Viruses are responsible for the majority of infectious diseases, from the common cold to HIV/AIDS or hemorrhagic fevers, the latter with devastating effects on the human population. Accordingly, the development of efficient antiviral therapies is a major goal and a challenge for the scientific community, as we are still far from understanding the molecular mechanisms that operate after virus infection. Interferon-stimulated gene 15 (ISG15) plays an important antiviral role during viral infection. ISG15 catalyzes a ubiquitin-like post-translational modification termed ISGylation, involving the conjugation of ISG15 molecules to de novo synthesized viral or cellular proteins, which regulates their stability and function. Numerous biomedically relevant viruses are targets of ISG15, as well as proteins involved in antiviral immunity. Beyond their role as cellular powerhouses, mitochondria are multifunctional organelles that act as signaling hubs in antiviral responses. In this review, we give an overview of the biological consequences of ISGylation for virus infection and host defense. We also compare several published proteomic studies to identify and classify potential mitochondrial ISGylation targets. Finally, based on our recent observations, we discuss the essential functions of mitochondria in the antiviral response and examine the role of ISG15 in the regulation of mitochondrial processes, specifically OXPHOS and mitophagy.

]]>ISG15, a Small Molecule with Huge Implications: Regulation of Mitochondrial HomeostasisManuel AlbertMartina BécaresMichela FalquiCarlos Fernández-LozanoSusana Guerradoi: 10.3390/v10110629Viruses2018-11-13Viruses2018-11-131011Review62910.3390/v10110629http://www.mdpi.com/1999-4915/10/11/629Viruses, Vol. 10, Pages 628: Detection of a Conspecific Mycovirus in Two Closely Related Native and Introduced Fungal Hosts and Evidence for Interspecific Virus Transmissionhttp://www.mdpi.com/1999-4915/10/11/628
Hymenoscyphus albidus is a native fungus in Europe where it behaves as a harmless decomposer of leaves of common ash. Its close relative Hymenoscyphus fraxineus was introduced into Europe from Asia and currently threatens ash (Fraxinus sp.) stands all across the continent causing ash dieback. H. fraxineus isolates from Europe were previously shown to harbor a mycovirus named Hymenoscyphus fraxineus Mitovirus 1 (HfMV1). In the present study, we describe a conspecific mycovirus that we detected in H. albidus. HfMV1 was consistently identified in H. albidus isolates (mean prevalence: 49.3%) which were collected in the sampling areas before the arrival of ash dieback. HfMV1 strains in both fungal hosts contain a single ORF of identical length (717 AA) for which a mean pairwise identity of 94.5% was revealed. The occurrence of a conspecific mitovirus in H. albidus and H. fraxineus is most likely the result of parallel virus evolution in the two fungal hosts. HfMV1 sequences from H. albidus showed a higher nucleotide diversity and a higher number of mutations compared to those from H. fraxineus, probably due to a bottleneck caused by the introduction of H. fraxineus in Europe. Our data also points to multiple interspecific virus transfers from H. albidus to H. fraxineus, which could have contributed to the intraspecific virus diversity found in H. fraxineus.Viruses, Vol. 10, Pages 628: Detection of a Conspecific Mycovirus in Two Closely Related Native and Introduced Fungal Hosts and Evidence for Interspecific Virus Transmission

Hymenoscyphus albidus is a native fungus in Europe where it behaves as a harmless decomposer of leaves of common ash. Its close relative Hymenoscyphus fraxineus was introduced into Europe from Asia and currently threatens ash (Fraxinus sp.) stands all across the continent causing ash dieback. H. fraxineus isolates from Europe were previously shown to harbor a mycovirus named Hymenoscyphus fraxineus Mitovirus 1 (HfMV1). In the present study, we describe a conspecific mycovirus that we detected in H. albidus. HfMV1 was consistently identified in H. albidus isolates (mean prevalence: 49.3%) which were collected in the sampling areas before the arrival of ash dieback. HfMV1 strains in both fungal hosts contain a single ORF of identical length (717 AA) for which a mean pairwise identity of 94.5% was revealed. The occurrence of a conspecific mitovirus in H. albidus and H. fraxineus is most likely the result of parallel virus evolution in the two fungal hosts. HfMV1 sequences from H. albidus showed a higher nucleotide diversity and a higher number of mutations compared to those from H. fraxineus, probably due to a bottleneck caused by the introduction of H. fraxineus in Europe. Our data also points to multiple interspecific virus transfers from H. albidus to H. fraxineus, which could have contributed to the intraspecific virus diversity found in H. fraxineus.

]]>Detection of a Conspecific Mycovirus in Two Closely Related Native and Introduced Fungal Hosts and Evidence for Interspecific Virus TransmissionCorine N. SchoebelSimone ProsperoAndrin GrossDaniel Riglingdoi: 10.3390/v10110628Viruses2018-11-13Viruses2018-11-131011Article62810.3390/v10110628http://www.mdpi.com/1999-4915/10/11/628Viruses, Vol. 10, Pages 627: Causes and Consequences of Spatial Within-Host Viral Spreadhttp://www.mdpi.com/1999-4915/10/11/627
The spread of viral pathogens both between and within hosts is inherently a spatial process. While the spatial aspects of viral spread at the epidemiological level have been increasingly well characterized, the spatial aspects of viral spread within infected hosts are still understudied. Here, with a focus on influenza A viruses (IAVs), we first review experimental studies that have shed light on the mechanisms and spatial dynamics of viral spread within hosts. These studies provide strong empirical evidence for highly localized IAV spread within hosts. Since mathematical and computational within-host models have been increasingly used to gain a quantitative understanding of observed viral dynamic patterns, we then review the (relatively few) computational modeling studies that have shed light on possible factors that structure the dynamics of spatial within-host IAV spread. These factors include the dispersal distance of virions, the localization of the immune response, and heterogeneity in host cell phenotypes across the respiratory tract. While informative, we find in these studies a striking absence of theoretical expectations of how spatial dynamics may impact the dynamics of viral populations. To mitigate this, we turn to the extensive ecological and evolutionary literature on range expansions to provide informed theoretical expectations. We find that factors such as the type of density dependence, the frequency of long-distance dispersal, specific life history characteristics, and the extent of spatial heterogeneity are critical factors affecting the speed of population spread and the genetic composition of spatially expanding populations. For each factor that we identified in the theoretical literature, we draw parallels to its analog in viral populations. We end by discussing current knowledge gaps related to the spatial component of within-host IAV spread and the potential for within-host spatial considerations to inform the development of disease control strategies.Viruses, Vol. 10, Pages 627: Causes and Consequences of Spatial Within-Host Viral Spread

The spread of viral pathogens both between and within hosts is inherently a spatial process. While the spatial aspects of viral spread at the epidemiological level have been increasingly well characterized, the spatial aspects of viral spread within infected hosts are still understudied. Here, with a focus on influenza A viruses (IAVs), we first review experimental studies that have shed light on the mechanisms and spatial dynamics of viral spread within hosts. These studies provide strong empirical evidence for highly localized IAV spread within hosts. Since mathematical and computational within-host models have been increasingly used to gain a quantitative understanding of observed viral dynamic patterns, we then review the (relatively few) computational modeling studies that have shed light on possible factors that structure the dynamics of spatial within-host IAV spread. These factors include the dispersal distance of virions, the localization of the immune response, and heterogeneity in host cell phenotypes across the respiratory tract. While informative, we find in these studies a striking absence of theoretical expectations of how spatial dynamics may impact the dynamics of viral populations. To mitigate this, we turn to the extensive ecological and evolutionary literature on range expansions to provide informed theoretical expectations. We find that factors such as the type of density dependence, the frequency of long-distance dispersal, specific life history characteristics, and the extent of spatial heterogeneity are critical factors affecting the speed of population spread and the genetic composition of spatially expanding populations. For each factor that we identified in the theoretical literature, we draw parallels to its analog in viral populations. We end by discussing current knowledge gaps related to the spatial component of within-host IAV spread and the potential for within-host spatial considerations to inform the development of disease control strategies.

]]>Causes and Consequences of Spatial Within-Host Viral SpreadMolly E. GallagherChristopher B. BrookeRuian KeKatia Koelledoi: 10.3390/v10110627Viruses2018-11-13Viruses2018-11-131011Review62710.3390/v10110627http://www.mdpi.com/1999-4915/10/11/627Viruses, Vol. 10, Pages 626: Ultrasensitive and Fast Diagnostics of Viable Listeria Cells by CBD Magnetic Separation Combined with A511::luxAB Detectionhttp://www.mdpi.com/1999-4915/10/11/626
The genus Listeria includes foodborne pathogens that cause life-threatening infections in those at risk, and sensitive and specific methods for detection of these bacteria are needed. Based on their unrivaled host specificity and ability to discriminate viable cells, bacteriophages represent an ideal toolbox for the development of such methods. Here, the authors describe an ultrasensitive diagnostic protocol for Listeria by combining two phage-based strategies: (1) specific capture and concentration of target cells by magnetic separation, harnessing cell wall-binding domains from Listeria phage endolysins (CBD-MS); and (2) highly sensitive detection using an adaptation of the A511::luxAB bioluminescent reporter phage assay in a microwell plate format. The combined assay enabled direct detection of approximately 100 bacteria per ml of pure culture with genus-level specificity in less than 6 h. For contaminated foods, the procedure included a 16 h selective enrichment step, followed by CBD-MS separation and A511::luxAB detection. It was able to consistently detect extremely low numbers (0.1 to 1.0 cfu/g) of viable Listeria cells, in a total assay time of less than 22 h. These results demonstrate the superiority of this phage-based assay to standard culture-based diagnostic protocols for the detection of viable bacteria, with respect to both sensitivity and speed.Viruses, Vol. 10, Pages 626: Ultrasensitive and Fast Diagnostics of Viable Listeria Cells by CBD Magnetic Separation Combined with A511::luxAB Detection

The genus Listeria includes foodborne pathogens that cause life-threatening infections in those at risk, and sensitive and specific methods for detection of these bacteria are needed. Based on their unrivaled host specificity and ability to discriminate viable cells, bacteriophages represent an ideal toolbox for the development of such methods. Here, the authors describe an ultrasensitive diagnostic protocol for Listeria by combining two phage-based strategies: (1) specific capture and concentration of target cells by magnetic separation, harnessing cell wall-binding domains from Listeria phage endolysins (CBD-MS); and (2) highly sensitive detection using an adaptation of the A511::luxAB bioluminescent reporter phage assay in a microwell plate format. The combined assay enabled direct detection of approximately 100 bacteria per ml of pure culture with genus-level specificity in less than 6 h. For contaminated foods, the procedure included a 16 h selective enrichment step, followed by CBD-MS separation and A511::luxAB detection. It was able to consistently detect extremely low numbers (0.1 to 1.0 cfu/g) of viable Listeria cells, in a total assay time of less than 22 h. These results demonstrate the superiority of this phage-based assay to standard culture-based diagnostic protocols for the detection of viable bacteria, with respect to both sensitivity and speed.

]]>Ultrasensitive and Fast Diagnostics of Viable Listeria Cells by CBD Magnetic Separation Combined with A511::luxAB DetectionJan W. KretzerMathias SchmelcherMartin J. Loessnerdoi: 10.3390/v10110626Viruses2018-11-13Viruses2018-11-131011Article62610.3390/v10110626http://www.mdpi.com/1999-4915/10/11/626Viruses, Vol. 10, Pages 625: Broad-Spectrum Antiviral Activity of an Ankyrin Repeat Protein on Viral Assembly against Chimeric NL4-3 Viruses Carrying Gag/PR Derived from Circulating Strains among Northern Thai Patientshttp://www.mdpi.com/1999-4915/10/11/625
Certain proteins have demonstrated proficient human immunodeficiency virus (HIV-1) life cycle disturbance. Recently, the ankyrin repeat protein targeting the HIV-1 capsid, AnkGAG1D4, showed a negative effect on the viral assembly of the HIV-1NL4-3 laboratory strain. To extend its potential for future clinical application, the activity of AnkGAG1D4 in the inhibition of other HIV-1 circulating strains was evaluated. Chimeric NL4-3 viruses carrying patient-derived Gag/PR-coding regions were generated from 131 antiretroviral drug-na&amp;iuml;ve HIV-1 infected individuals in northern Thailand during 2001&amp;ndash;2012. SupT1, a stable T-cell line expressing AnkGAG1D4 and ankyrin non-binding control (AnkA32D3), were challenged with these chimeric viruses. The p24CA sequences were analysed and classified using the K-means clustering method. Among all the classes of virus classified using the p24CA sequences, SupT1/AnkGAG1D4 demonstrated significantly lower levels of p24CA than SupT1/AnkA32D3, which was found to correlate with the syncytia formation. This result suggests that AnkGAG1D4 can significantly interfere with the chimeric viruses derived from patients with different sequences of the p24CA domain. It supports the possibility of ankyrin-based therapy as a broad alternative therapeutic molecule for HIV-1 gene therapy in the future.Viruses, Vol. 10, Pages 625: Broad-Spectrum Antiviral Activity of an Ankyrin Repeat Protein on Viral Assembly against Chimeric NL4-3 Viruses Carrying Gag/PR Derived from Circulating Strains among Northern Thai Patients

Certain proteins have demonstrated proficient human immunodeficiency virus (HIV-1) life cycle disturbance. Recently, the ankyrin repeat protein targeting the HIV-1 capsid, AnkGAG1D4, showed a negative effect on the viral assembly of the HIV-1NL4-3 laboratory strain. To extend its potential for future clinical application, the activity of AnkGAG1D4 in the inhibition of other HIV-1 circulating strains was evaluated. Chimeric NL4-3 viruses carrying patient-derived Gag/PR-coding regions were generated from 131 antiretroviral drug-na&amp;iuml;ve HIV-1 infected individuals in northern Thailand during 2001&amp;ndash;2012. SupT1, a stable T-cell line expressing AnkGAG1D4 and ankyrin non-binding control (AnkA32D3), were challenged with these chimeric viruses. The p24CA sequences were analysed and classified using the K-means clustering method. Among all the classes of virus classified using the p24CA sequences, SupT1/AnkGAG1D4 demonstrated significantly lower levels of p24CA than SupT1/AnkA32D3, which was found to correlate with the syncytia formation. This result suggests that AnkGAG1D4 can significantly interfere with the chimeric viruses derived from patients with different sequences of the p24CA domain. It supports the possibility of ankyrin-based therapy as a broad alternative therapeutic molecule for HIV-1 gene therapy in the future.

We present the recently isolated virus vB_BthP-Goe4 infecting Bacillus thuringiensis HD1. Morphological investigation via transmission electron microscopy revealed key characteristics of the genus Phi29virus, but with an elongated head resulting in larger virion particles of approximately 50 nm width and 120 nm height. Genome sequencing and analysis resulted in a linear phage chromosome of approximately 26 kb, harbouring 40 protein-encoding genes and a packaging RNA. Sequence comparison confirmed the relation to the Phi29virus genus and genomes of other related strains. A global average nucleotide identity analysis of all identified &amp;phi;29-like viruses revealed the formation of several new groups previously not observed. The largest group includes Goe4 and may significantly expand the genus Phi29virus (Salasvirus) or the Picovirinae subfamily.

]]>Genomic Analysis of the Recent Viral Isolate vB_BthP-Goe4 Reveals Increased Diversity of φ29-Like PhagesTobias SchillingMichael HoppertRobert Herteldoi: 10.3390/v10110624Viruses2018-11-13Viruses2018-11-131011Article62410.3390/v10110624http://www.mdpi.com/1999-4915/10/11/624Viruses, Vol. 10, Pages 623: Following in the Footsteps of the Chikungunya Virus in Brazil: The First Autochthonous Cases in Amapá in 2014 and Its Emergence in Rio de Janeiro during 2016http://www.mdpi.com/1999-4915/10/11/623
Currently, Brazil lives a triple arboviruses epidemic (DENV, ZIKV and CHIKV) making the differential diagnosis difficult for health professionals. Here, we aimed to investigate chikungunya cases and the possible occurrence of co-infections during the epidemic in Amap&amp;aacute; (AP) that started in 2014 when the first autochthonous cases were reported and in Rio de Janeiro (RJ) in 2016. We further performed molecular characterization and genotyping of representative strains. In AP, 51.4% of the suspected cases were confirmed for CHIKV, 71.0% (76/107). Of those, 24 co-infections by CHIKV/DENV, two by CHIKV/DENV-1, and two by CHIKV/DENV-4 were observed. In RJ, 76.9% of the suspected cases were confirmed for CHIKV and co-infections by CHIKV/DENV (n = 8) and by CHIKV/ZIKV (n = 17) were observed. Overall, fever, arthralgia, myalgia, prostration, edema, exanthema, conjunctival hyperemia, lower back pain, dizziness, nausea, retroorbital pain, and anorexia were the predominating chikungunya clinical symptoms described. All strains analyzed from AP belonged to the Asian genotype and no amino acid changes were observed. In RJ, the East-Central-South-African genotype (ECSA) circulation was demonstrated and no E1-A226V mutation was observed. Despite this, an E1-V156A substitution was characterized in two samples and for the first time, the E1-K211T mutation was reported in all samples analyzed.Viruses, Vol. 10, Pages 623: Following in the Footsteps of the Chikungunya Virus in Brazil: The First Autochthonous Cases in Amapá in 2014 and Its Emergence in Rio de Janeiro during 2016

Currently, Brazil lives a triple arboviruses epidemic (DENV, ZIKV and CHIKV) making the differential diagnosis difficult for health professionals. Here, we aimed to investigate chikungunya cases and the possible occurrence of co-infections during the epidemic in Amap&amp;aacute; (AP) that started in 2014 when the first autochthonous cases were reported and in Rio de Janeiro (RJ) in 2016. We further performed molecular characterization and genotyping of representative strains. In AP, 51.4% of the suspected cases were confirmed for CHIKV, 71.0% (76/107). Of those, 24 co-infections by CHIKV/DENV, two by CHIKV/DENV-1, and two by CHIKV/DENV-4 were observed. In RJ, 76.9% of the suspected cases were confirmed for CHIKV and co-infections by CHIKV/DENV (n = 8) and by CHIKV/ZIKV (n = 17) were observed. Overall, fever, arthralgia, myalgia, prostration, edema, exanthema, conjunctival hyperemia, lower back pain, dizziness, nausea, retroorbital pain, and anorexia were the predominating chikungunya clinical symptoms described. All strains analyzed from AP belonged to the Asian genotype and no amino acid changes were observed. In RJ, the East-Central-South-African genotype (ECSA) circulation was demonstrated and no E1-A226V mutation was observed. Despite this, an E1-V156A substitution was characterized in two samples and for the first time, the E1-K211T mutation was reported in all samples analyzed.

]]>Following in the Footsteps of the Chikungunya Virus in Brazil: The First Autochthonous Cases in Amapá in 2014 and Its Emergence in Rio de Janeiro during 2016Thiara Manuele Alves de SouzaEdcelha D’Athaide RibeiroValmir Corrêa e CorrêaPaulo Vieira DamascoCarla Cunha SantosFernanda de Bruycker-NogueiraThaís Chouin-CarneiroNieli Rodrigues da Costa FariaPriscila Conrado Guerra NunesManoela HeringerMonique da Rocha Queiroz LimaJéssica Badolato-CorrêaMárcio da Costa CipitelliElzinandes Leal de AzeredoRita Maria Ribeiro NogueiraFlavia Barreto dos Santosdoi: 10.3390/v10110623Viruses2018-11-12Viruses2018-11-121011Article62310.3390/v10110623http://www.mdpi.com/1999-4915/10/11/623Viruses, Vol. 10, Pages 622: Antibiotic Therapy Using Phage Depolymerases: Robustness Across a Range of Conditionshttp://www.mdpi.com/1999-4915/10/11/622
Phage-derived depolymerases directed against bacterial capsules are showing therapeutic promise in various animal models of infection. However, individual animal model studies are often constrained by use of highly specific protocols, such that results may not generalize to even slight modifications. Here we explore the robustness of depolymerase therapies shown to succeed in a previous study of mice. Treatment success rates were reduced by treatment delay, more so for some enzymes than others: K1- and K5 capsule-degrading enzymes retained partial efficacy on delay, while K30 depolymerase did not. Phage were superior to enzymes under delayed treatment only for K1. Route of administration (intramuscular versus intraperitoneal) mattered for success of K1E, possibly for K1F, not for K1H depolymerase. Significantly, K1 capsule-degrading enzymes proved highly successful when using immune-suppressed, leukopenic mice, even with delayed treatment. Evolution of bacteria resistant to K1-degrading enzymes did not thwart therapeutic success in leukopenic mice, likely because resistant bacteria were avirulent. In combination with previous studies these results continue to support the efficacy of depolymerases as antibacterial agents in vivo, but system-specific details are becoming evident.Viruses, Vol. 10, Pages 622: Antibiotic Therapy Using Phage Depolymerases: Robustness Across a Range of Conditions

Phage-derived depolymerases directed against bacterial capsules are showing therapeutic promise in various animal models of infection. However, individual animal model studies are often constrained by use of highly specific protocols, such that results may not generalize to even slight modifications. Here we explore the robustness of depolymerase therapies shown to succeed in a previous study of mice. Treatment success rates were reduced by treatment delay, more so for some enzymes than others: K1- and K5 capsule-degrading enzymes retained partial efficacy on delay, while K30 depolymerase did not. Phage were superior to enzymes under delayed treatment only for K1. Route of administration (intramuscular versus intraperitoneal) mattered for success of K1E, possibly for K1F, not for K1H depolymerase. Significantly, K1 capsule-degrading enzymes proved highly successful when using immune-suppressed, leukopenic mice, even with delayed treatment. Evolution of bacteria resistant to K1-degrading enzymes did not thwart therapeutic success in leukopenic mice, likely because resistant bacteria were avirulent. In combination with previous studies these results continue to support the efficacy of depolymerases as antibacterial agents in vivo, but system-specific details are becoming evident.

]]>Antibiotic Therapy Using Phage Depolymerases: Robustness Across a Range of ConditionsHan LinMatthew L. PaffIan J. MolineuxJames J. Bulldoi: 10.3390/v10110622Viruses2018-11-12Viruses2018-11-121011Article62210.3390/v10110622http://www.mdpi.com/1999-4915/10/11/622Viruses, Vol. 10, Pages 621: Unlocking the Potential of 46 New Bacteriophages for Biocontrol of Dickeya Solanihttp://www.mdpi.com/1999-4915/10/11/621
Modern agriculture is expected to face an increasing global demand for food while also needing to comply with higher sustainability standards. Therefore, control of crop pathogens requires new, green alternatives to current methods. Potatoes are susceptible to several bacterial diseases, with infections by soft rot Enterobacteriaceae (SRE) being a significant contributor to the major annual losses. As there are currently no efficient ways of combating SRE, we sought to develop an approach that could easily be incorporated into the potato production pipeline. To this end, 46 phages infecting the emerging potato pathogen Dickeya solani were isolated and thoroughly characterized. The 46 isolated phages were grouped into three different groups based on DNA similarity, representing two distinct clusters and a singleton. One cluster showed similarity to phages previously used to successfully treat soft rot in potatoes, whereas the remaining phages were novel and showed only very limited similarity to previously isolated phages. We selected six diverse phages in order to create the hereto most complex phage cocktail against SRE. The cocktail was applied in a proof-of-principle experiment to treat soft rot in potatoes under simulated storage conditions. We show that the phage cocktail was able to significantly reduce the incidence of soft rot as well as disease severity after 5 days of storage post-infection with Dickeya solani. This confirms results from previous studies that phages represent promising biocontrol agents against SRE infection in potato.Viruses, Vol. 10, Pages 621: Unlocking the Potential of 46 New Bacteriophages for Biocontrol of Dickeya Solani

Modern agriculture is expected to face an increasing global demand for food while also needing to comply with higher sustainability standards. Therefore, control of crop pathogens requires new, green alternatives to current methods. Potatoes are susceptible to several bacterial diseases, with infections by soft rot Enterobacteriaceae (SRE) being a significant contributor to the major annual losses. As there are currently no efficient ways of combating SRE, we sought to develop an approach that could easily be incorporated into the potato production pipeline. To this end, 46 phages infecting the emerging potato pathogen Dickeya solani were isolated and thoroughly characterized. The 46 isolated phages were grouped into three different groups based on DNA similarity, representing two distinct clusters and a singleton. One cluster showed similarity to phages previously used to successfully treat soft rot in potatoes, whereas the remaining phages were novel and showed only very limited similarity to previously isolated phages. We selected six diverse phages in order to create the hereto most complex phage cocktail against SRE. The cocktail was applied in a proof-of-principle experiment to treat soft rot in potatoes under simulated storage conditions. We show that the phage cocktail was able to significantly reduce the incidence of soft rot as well as disease severity after 5 days of storage post-infection with Dickeya solani. This confirms results from previous studies that phages represent promising biocontrol agents against SRE infection in potato.

]]>Unlocking the Potential of 46 New Bacteriophages for Biocontrol of Dickeya SolaniAlexander B. CarstensAmaru M. DjurhuusWitold KotDeborah Jacobs-SeraGraham F. HatfullLars H. Hansendoi: 10.3390/v10110621Viruses2018-11-10Viruses2018-11-101011Article62110.3390/v10110621http://www.mdpi.com/1999-4915/10/11/621Viruses, Vol. 10, Pages 620: Detailed Characterization of Early HIV-1 Replication Dynamics in Primary Human Macrophageshttp://www.mdpi.com/1999-4915/10/11/620
Macrophages are natural target cells of human immunodeficiency virus type 1 (HIV-1). Viral replication appears to be delayed in these cells compared to lymphocytes; however, little is known about the kinetics of early post-entry events. Time-of-addition experiments using several HIV-1 inhibitors and the detection of reverse transcriptase (RT) products with droplet digital PCR (ddPCR) revealed that early replication was delayed in primary human monocyte-derived macrophages of several donors and peaked late after infection. Direct imaging of reverse-transcription and pre-integration complexes (RTC/PIC) by click-labeling of newly synthesized DNA further confirmed our findings and showed a concomitant shift to the nuclear stage over time. Altering the entry pathway enhanced infectivity but did not affect kinetics of viral replication. The addition of viral protein X (Vpx) enhanced productive infection and accelerated completion of reverse transcription and nuclear entry. We propose that sterile alpha motif (SAM) and histidine/aspartate (HD) domain-containing protein 1 (SAMHD1) activity lowering deoxyribonucleotide triphosphate (dNTP) pools is the principal factor delaying early HIV-1 replication in macrophages.Viruses, Vol. 10, Pages 620: Detailed Characterization of Early HIV-1 Replication Dynamics in Primary Human Macrophages

Macrophages are natural target cells of human immunodeficiency virus type 1 (HIV-1). Viral replication appears to be delayed in these cells compared to lymphocytes; however, little is known about the kinetics of early post-entry events. Time-of-addition experiments using several HIV-1 inhibitors and the detection of reverse transcriptase (RT) products with droplet digital PCR (ddPCR) revealed that early replication was delayed in primary human monocyte-derived macrophages of several donors and peaked late after infection. Direct imaging of reverse-transcription and pre-integration complexes (RTC/PIC) by click-labeling of newly synthesized DNA further confirmed our findings and showed a concomitant shift to the nuclear stage over time. Altering the entry pathway enhanced infectivity but did not affect kinetics of viral replication. The addition of viral protein X (Vpx) enhanced productive infection and accelerated completion of reverse transcription and nuclear entry. We propose that sterile alpha motif (SAM) and histidine/aspartate (HD) domain-containing protein 1 (SAMHD1) activity lowering deoxyribonucleotide triphosphate (dNTP) pools is the principal factor delaying early HIV-1 replication in macrophages.

]]>Detailed Characterization of Early HIV-1 Replication Dynamics in Primary Human MacrophagesDavid Alejandro BejaranoMaria C. PuertasKathleen BörnerJavier Martinez-PicadoBarbara MüllerHans-Georg Kräusslichdoi: 10.3390/v10110620Viruses2018-11-10Viruses2018-11-101011Article62010.3390/v10110620http://www.mdpi.com/1999-4915/10/11/620Viruses, Vol. 10, Pages 619: Inter- and Intra-Host Nucleotide Variations in Hepatitis A Virus in Culture and Clinical Samples Detected by Next-Generation Sequencinghttp://www.mdpi.com/1999-4915/10/11/619
The accurate virus detection, strain discrimination, and source attribution of contaminated food items remains a persistent challenge because of the high mutation rates anticipated to occur in foodborne RNA viruses, such as hepatitis A virus (HAV). This has led to predictions of the existence of more than one sequence variant between the hosts (inter-host) or within an individual host (intra-host). However, there have been no reports of intra-host variants from an infected single individual, and little is known about the accuracy of the single nucleotide variations (SNVs) calling with various methods. In this study, the presence and identity of viral SNVs, either between HAV clinical specimens or among a series of samples derived from HAV clone1-infected FRhK4 cells, were determined following analyses of nucleotide sequences generated using next-generation sequencing (NGS) and pyrosequencing methods. The results demonstrate the co-existence of inter- and intra-host variants both in the clinical specimens and the cultured samples. The discovery and confirmation of multi-viral RNAs in an infected individual is dependent on the strain discrimination at the SNV level, and critical for successful outbreak traceback and source attribution investigations. The detection of SNVs in a time series of HAV infected FRhK4 cells improved our understanding on the mutation dynamics determined probably by different selective pressures. Additionally, it demonstrated that NGS could potentially provide a valuable investigative approach toward SNV detection and identification for other RNA viruses.Viruses, Vol. 10, Pages 619: Inter- and Intra-Host Nucleotide Variations in Hepatitis A Virus in Culture and Clinical Samples Detected by Next-Generation Sequencing

The accurate virus detection, strain discrimination, and source attribution of contaminated food items remains a persistent challenge because of the high mutation rates anticipated to occur in foodborne RNA viruses, such as hepatitis A virus (HAV). This has led to predictions of the existence of more than one sequence variant between the hosts (inter-host) or within an individual host (intra-host). However, there have been no reports of intra-host variants from an infected single individual, and little is known about the accuracy of the single nucleotide variations (SNVs) calling with various methods. In this study, the presence and identity of viral SNVs, either between HAV clinical specimens or among a series of samples derived from HAV clone1-infected FRhK4 cells, were determined following analyses of nucleotide sequences generated using next-generation sequencing (NGS) and pyrosequencing methods. The results demonstrate the co-existence of inter- and intra-host variants both in the clinical specimens and the cultured samples. The discovery and confirmation of multi-viral RNAs in an infected individual is dependent on the strain discrimination at the SNV level, and critical for successful outbreak traceback and source attribution investigations. The detection of SNVs in a time series of HAV infected FRhK4 cells improved our understanding on the mutation dynamics determined probably by different selective pressures. Additionally, it demonstrated that NGS could potentially provide a valuable investigative approach toward SNV detection and identification for other RNA viruses.

]]>Inter- and Intra-Host Nucleotide Variations in Hepatitis A Virus in Culture and Clinical Samples Detected by Next-Generation SequencingZhihui YangMark MammelChris A. WhitehouseDiana NgoMichael Kulkadoi: 10.3390/v10110619Viruses2018-11-09Viruses2018-11-091011Article61910.3390/v10110619http://www.mdpi.com/1999-4915/10/11/619Viruses, Vol. 10, Pages 618: CMV2b-Dependent Regulation of Host Defense Pathways in the Context of Viral Infectionhttp://www.mdpi.com/1999-4915/10/11/618
RNA silencing (or RNA interference, RNAi) plays direct roles in plant host defenses against viruses. Viruses encode suppressors of RNAi (VSRs) to counteract host antiviral defenses. The generation of transgenic plants expressing VSRs facilitates the understanding of the mechanisms of VSR-mediated interference with the endogenous silencing pathway. However, studying VSRs independent of other viral components simplifies the complex roles of VSRs during natural viral infection. While suppression of transgene silencing by the VSR 2b protein encoded by cucumber mosaic virus (CMV) requires 2b-small RNA (sRNA) binding activity, suppression of host antiviral defenses requires the binding activity of both sRNAs and AGOs proteins. This study, aimed to understand the functions of 2b in the context of CMV infection; thus, we performed genome-wide analyses of differential DNA methylation regions among wild-type CMV-infected, CMV&amp;Delta;2b-infected, and 2b-transgenic Arabidopsis plants. These analyses, together with transcriptome sequencing and RT-qPCR analyses, show that while the majority of induced genes in 2b-transgenic plants were involved in extensive metabolic pathways, CMV-infection 2b-dependent induced genes were enriched in plant immunity pathways, including salicylic acid (SA) signaling. Together with infection with CMV mutants that expressed the 2b functional domains of sRNA or AGO binding, our data demonstrate that CMV-accelerated SA signaling depends on 2b-sRNA binding activity which is also responsible for virulence.Viruses, Vol. 10, Pages 618: CMV2b-Dependent Regulation of Host Defense Pathways in the Context of Viral Infection

RNA silencing (or RNA interference, RNAi) plays direct roles in plant host defenses against viruses. Viruses encode suppressors of RNAi (VSRs) to counteract host antiviral defenses. The generation of transgenic plants expressing VSRs facilitates the understanding of the mechanisms of VSR-mediated interference with the endogenous silencing pathway. However, studying VSRs independent of other viral components simplifies the complex roles of VSRs during natural viral infection. While suppression of transgene silencing by the VSR 2b protein encoded by cucumber mosaic virus (CMV) requires 2b-small RNA (sRNA) binding activity, suppression of host antiviral defenses requires the binding activity of both sRNAs and AGOs proteins. This study, aimed to understand the functions of 2b in the context of CMV infection; thus, we performed genome-wide analyses of differential DNA methylation regions among wild-type CMV-infected, CMV&amp;Delta;2b-infected, and 2b-transgenic Arabidopsis plants. These analyses, together with transcriptome sequencing and RT-qPCR analyses, show that while the majority of induced genes in 2b-transgenic plants were involved in extensive metabolic pathways, CMV-infection 2b-dependent induced genes were enriched in plant immunity pathways, including salicylic acid (SA) signaling. Together with infection with CMV mutants that expressed the 2b functional domains of sRNA or AGO binding, our data demonstrate that CMV-accelerated SA signaling depends on 2b-sRNA binding activity which is also responsible for virulence.

]]>CMV2b-Dependent Regulation of Host Defense Pathways in the Context of Viral InfectionJian-Hua ZhaoXiao-Lan LiuYuan-Yuan FangRong-Xiang FangHui-Shan Guodoi: 10.3390/v10110618Viruses2018-11-09Viruses2018-11-091011Article61810.3390/v10110618http://www.mdpi.com/1999-4915/10/11/618Viruses, Vol. 10, Pages 617: Effects of Staphylococcus aureus Bacteriophage K on Expression of Cytokines and Activation Markers by Human Dendritic Cells In Vitrohttp://www.mdpi.com/1999-4915/10/11/617
A potential concern with bacteriophage (phage) therapeutics is a host-versus-phage response in which the immune system may neutralize or destroy phage particles and thus impair therapeutic efficacy, or a strong inflammatory response to repeated phage exposure might endanger the patient. Current literature is discrepant with regard to the nature and magnitude of innate and adaptive immune response to phages. The purpose of this work was to study the potential effects of Staphylococcus aureus phage K on the activation of human monocyte-derived dendritic cells. Since phage K acquired from ATCC was isolated around 90 years ago, we first tested its activity against a panel of 36 diverse S. aureus clinical isolates from military patients and found that it was lytic against 30/36 (83%) of strains. Human monocyte-derived dendritic cells were used to test for an in vitro phage-specific inflammatory response. Repeated experiments demonstrated that phage K had little impact on the expression of pro- and anti-inflammatory cytokines, or on MHC-I/II and CD80/CD86 protein expression. Given that dendritic cells are potent antigen-presenting cells and messengers between the innate and the adaptive immune systems, our results suggest that phage K does not independently affect cellular immunity or has a very limited impact on it.Viruses, Vol. 10, Pages 617: Effects of Staphylococcus aureus Bacteriophage K on Expression of Cytokines and Activation Markers by Human Dendritic Cells In Vitro

A potential concern with bacteriophage (phage) therapeutics is a host-versus-phage response in which the immune system may neutralize or destroy phage particles and thus impair therapeutic efficacy, or a strong inflammatory response to repeated phage exposure might endanger the patient. Current literature is discrepant with regard to the nature and magnitude of innate and adaptive immune response to phages. The purpose of this work was to study the potential effects of Staphylococcus aureus phage K on the activation of human monocyte-derived dendritic cells. Since phage K acquired from ATCC was isolated around 90 years ago, we first tested its activity against a panel of 36 diverse S. aureus clinical isolates from military patients and found that it was lytic against 30/36 (83%) of strains. Human monocyte-derived dendritic cells were used to test for an in vitro phage-specific inflammatory response. Repeated experiments demonstrated that phage K had little impact on the expression of pro- and anti-inflammatory cytokines, or on MHC-I/II and CD80/CD86 protein expression. Given that dendritic cells are potent antigen-presenting cells and messengers between the innate and the adaptive immune systems, our results suggest that phage K does not independently affect cellular immunity or has a very limited impact on it.

]]>Effects of Staphylococcus aureus Bacteriophage K on Expression of Cytokines and Activation Markers by Human Dendritic Cells In VitroHelen FreybergerYunxiu HeAmanda RothMikeljon NikolichAndrey Filippovdoi: 10.3390/v10110617Viruses2018-11-08Viruses2018-11-081011Article61710.3390/v10110617http://www.mdpi.com/1999-4915/10/11/617Viruses, Vol. 10, Pages 616: Characterization of the Escherichia coli Virulent Myophage ST32http://www.mdpi.com/1999-4915/10/11/616
The virulent phage ST32 that infects the Escherichia coli strain ST130 was isolated from a wastewater sample in China and analyzed. Morphological observations showed that phage ST32 belongs to the Myoviridae family, as it has an icosahedral capsid and long contractile tail. Host range analysis showed that it exhibits a broad range of hosts including non-pathogenic and pathogenic E. coli strains. Interestingly, phage ST32 had a much larger burst size when amplified at 20 &amp;deg;C as compared to 30 &amp;deg;C or 37 &amp;deg;C. Its double-stranded DNA genome was sequenced and found to contain 53,092 bp with a GC content of 44.14%. Seventy-nine open reading frames (ORFs) were identified and annotated as well as a tRNA-Arg. Only nineteen ORFs were assigned putative functions. A phylogenetic tree using the large terminase subunit revealed a close relatedness with four unclassified Myoviridae phages. A comparative genomic analysis of these phages showed that the Enterobacteria phage phiEcoM-GJ1 is the closest relative to ST32 and shares the same new branch in the phylogenetic tree. Still, these two phages share only 47 of 79 ORFs with more than 90% identity. Phage ST32 has unique characteristics that make it a potential biological control agent under specific conditions.Viruses, Vol. 10, Pages 616: Characterization of the Escherichia coli Virulent Myophage ST32

The virulent phage ST32 that infects the Escherichia coli strain ST130 was isolated from a wastewater sample in China and analyzed. Morphological observations showed that phage ST32 belongs to the Myoviridae family, as it has an icosahedral capsid and long contractile tail. Host range analysis showed that it exhibits a broad range of hosts including non-pathogenic and pathogenic E. coli strains. Interestingly, phage ST32 had a much larger burst size when amplified at 20 &amp;deg;C as compared to 30 &amp;deg;C or 37 &amp;deg;C. Its double-stranded DNA genome was sequenced and found to contain 53,092 bp with a GC content of 44.14%. Seventy-nine open reading frames (ORFs) were identified and annotated as well as a tRNA-Arg. Only nineteen ORFs were assigned putative functions. A phylogenetic tree using the large terminase subunit revealed a close relatedness with four unclassified Myoviridae phages. A comparative genomic analysis of these phages showed that the Enterobacteria phage phiEcoM-GJ1 is the closest relative to ST32 and shares the same new branch in the phylogenetic tree. Still, these two phages share only 47 of 79 ORFs with more than 90% identity. Phage ST32 has unique characteristics that make it a potential biological control agent under specific conditions.

]]>Characterization of the Escherichia coli Virulent Myophage ST32Honghui LiuHany GeageaGeneviève M. RousseauSimon J. LabrieDenise M. TremblayXinchun LiuSylvain Moineaudoi: 10.3390/v10110616Viruses2018-11-07Viruses2018-11-071011Article61610.3390/v10110616http://www.mdpi.com/1999-4915/10/11/616Viruses, Vol. 10, Pages 615: Persistence and Intra-Host Genetic Evolution of Zika Virus Infection in Symptomatic Adults: A Special View in the Male Reproductive Systemhttp://www.mdpi.com/1999-4915/10/11/615
We followed the presence of Zika virus (ZIKV) in four healthy adults (two men and two women), for periods ranging from 78 to 298 days post symptom onset. The patients were evaluated regarding the presence of the virus in different body fluids (blood, saliva, urine and semen), development of immune responses (including antibodies, cytokines and chemokines), and virus genetic variation within samples collected from semen and urine during the infection course. The analysis was focused primarily on the two male patients who shed the virus for up to 158 days after the initial symptoms. ZIKV particles were detected in the spermatozoa cytoplasm and flagella, in immature sperm cells and could also be isolated from semen in cell culture, confirming that the virus is able to preserve integrity and infectivity during replication in the male reproductive system (MRS). Despite the damage caused by ZIKV infection within the MRS, our data showed that ZIKV infection did not result in infertility at least in one of the male patients. This patient was able to conceive a child after the infection. We also detected alterations in the male genital cytokine milieu, which could play an important role in the replication and transmission of the virus which could considerably increase the risk of ZIKV sexual spread. In addition, full genome ZIKV sequences were obtained from several samples (mainly semen), which allowed us to monitor the evolution of the virus within a patient during the infection course. We observed genetic changes over time in consensus sequences and lower frequency intra-host single nucleotide variants (iSNV), that suggested independent compartmentalization of ZIKV populations in the reproductive and urinary systems. Altogether, the present observations confirm the risks associated with the long-term replication and shedding of ZIKV in the MRS and help to elucidate patterns of intra-host genetic evolution during long term replication of the virus.Viruses, Vol. 10, Pages 615: Persistence and Intra-Host Genetic Evolution of Zika Virus Infection in Symptomatic Adults: A Special View in the Male Reproductive System

We followed the presence of Zika virus (ZIKV) in four healthy adults (two men and two women), for periods ranging from 78 to 298 days post symptom onset. The patients were evaluated regarding the presence of the virus in different body fluids (blood, saliva, urine and semen), development of immune responses (including antibodies, cytokines and chemokines), and virus genetic variation within samples collected from semen and urine during the infection course. The analysis was focused primarily on the two male patients who shed the virus for up to 158 days after the initial symptoms. ZIKV particles were detected in the spermatozoa cytoplasm and flagella, in immature sperm cells and could also be isolated from semen in cell culture, confirming that the virus is able to preserve integrity and infectivity during replication in the male reproductive system (MRS). Despite the damage caused by ZIKV infection within the MRS, our data showed that ZIKV infection did not result in infertility at least in one of the male patients. This patient was able to conceive a child after the infection. We also detected alterations in the male genital cytokine milieu, which could play an important role in the replication and transmission of the virus which could considerably increase the risk of ZIKV sexual spread. In addition, full genome ZIKV sequences were obtained from several samples (mainly semen), which allowed us to monitor the evolution of the virus within a patient during the infection course. We observed genetic changes over time in consensus sequences and lower frequency intra-host single nucleotide variants (iSNV), that suggested independent compartmentalization of ZIKV populations in the reproductive and urinary systems. Altogether, the present observations confirm the risks associated with the long-term replication and shedding of ZIKV in the MRS and help to elucidate patterns of intra-host genetic evolution during long term replication of the virus.

Human cytomegalovirus (HCMV) is a ubiquitous herpesvirus for which there is no vaccine or cure. This viral infection, once acquired, is life-long, residing latently in hematopoietic cells. However, latently infected individuals with weakened immune systems often undergo HCMV reactivation, which can cause serious complications in immunosuppressed and immunocompromised patients. Current anti-viral therapies target late stages of viral replication, and are often met with therapeutic resistance, necessitating the development of novel therapeutics. In this current study, we identified a naturally-occurring flavonoid compound, deguelin, which inhibits HCMV lytic replication. Our findings reveal that nanomolar concentrations of deguelin significantly suppress the production of the infectious virus. Further, we show that deguelin inhibits the lytic cycle during the phase of the replication cycle consistent with early (E) gene and protein expression. Importantly, our data reveal that deguelin inhibits replication of a ganciclovir-resistant strain of HCMV. Together, our findings identify a novel, naturally occurring compound that may prove useful in the treatment of HCMV replication.

]]>The Natural Flavonoid Compound Deguelin Inhibits HCMV Lytic Replication within FibroblastsMasatoshi NukuiChristine M. O’ConnorEain A. Murphydoi: 10.3390/v10110614Viruses2018-11-07Viruses2018-11-071011Article61410.3390/v10110614http://www.mdpi.com/1999-4915/10/11/614Viruses, Vol. 10, Pages 613: Meeting Review: 2018 International Workshop on Structure and Function of the Lentiviral gp41 Cytoplasmic Tailhttp://www.mdpi.com/1999-4915/10/11/613
Recent developments in defining the role of the lentiviral envelope glycoprotein (Env) cytoplasmic tail (CT) in Env trafficking and incorporation into virus particles have advanced our understanding of viral replication and transmission. To stimulate additional progress in this field, the two-day International Workshop on Structure and Function of the Lentiviral gp41 Cytoplasmic Tail, co-organized by Eric Freed and James Hoxie, was held at the National Cancer Institute in Frederick, MD (26&amp;ndash;27 April 2018). The meeting served to bring together experts focused on the role of gp41 in HIV replication and to discuss the emerging mechanisms of CT-dependent trafficking, Env conformation and structure, host protein interaction, incorporation, and viral transmission. The conference was organized around the following three main hot topics in gp41 research: the role of host factors in CT-dependent Env incorporation, Env structure, and CT-mediated trafficking and transmission. This review highlights important topics and the advances in gp41 research that were discussed during the conference.Viruses, Vol. 10, Pages 613: Meeting Review: 2018 International Workshop on Structure and Function of the Lentiviral gp41 Cytoplasmic Tail

Recent developments in defining the role of the lentiviral envelope glycoprotein (Env) cytoplasmic tail (CT) in Env trafficking and incorporation into virus particles have advanced our understanding of viral replication and transmission. To stimulate additional progress in this field, the two-day International Workshop on Structure and Function of the Lentiviral gp41 Cytoplasmic Tail, co-organized by Eric Freed and James Hoxie, was held at the National Cancer Institute in Frederick, MD (26&amp;ndash;27 April 2018). The meeting served to bring together experts focused on the role of gp41 in HIV replication and to discuss the emerging mechanisms of CT-dependent trafficking, Env conformation and structure, host protein interaction, incorporation, and viral transmission. The conference was organized around the following three main hot topics in gp41 research: the role of host factors in CT-dependent Env incorporation, Env structure, and CT-mediated trafficking and transmission. This review highlights important topics and the advances in gp41 research that were discussed during the conference.

]]>Meeting Review: 2018 International Workshop on Structure and Function of the Lentiviral gp41 Cytoplasmic TailMelissa V. FernandezEric O. Freeddoi: 10.3390/v10110613Viruses2018-11-07Viruses2018-11-071011Meeting Report61310.3390/v10110613http://www.mdpi.com/1999-4915/10/11/613Viruses, Vol. 10, Pages 612: Viroid Diseases in Pome and Stone Fruit Trees and Koch’s Postulates: A Critical Assessmenthttp://www.mdpi.com/1999-4915/10/11/612
Composed of a naked circular non-protein-coding genomic RNA, counting only a few hundred nucleotides, viroids&amp;mdash;the smallest infectious agents known so far&amp;mdash;are able to replicate and move systemically in herbaceous and woody host plants, which concomitantly may develop specific diseases or remain symptomless. Several viroids have been reported to naturally infect pome and stone fruit trees, showing symptoms on leaves, fruits and/or bark. However, Koch&amp;rsquo;s postulates required for establishing on firm grounds the viroid etiology of these diseases, have not been met in all instances. Here, pome and stone fruit tree diseases, conclusively proven to be caused by viroids, are reviewed, and the need to pay closer attention to fulfilling Koch&amp;rsquo;s postulates is emphasized.Viruses, Vol. 10, Pages 612: Viroid Diseases in Pome and Stone Fruit Trees and Koch’s Postulates: A Critical Assessment

Composed of a naked circular non-protein-coding genomic RNA, counting only a few hundred nucleotides, viroids&amp;mdash;the smallest infectious agents known so far&amp;mdash;are able to replicate and move systemically in herbaceous and woody host plants, which concomitantly may develop specific diseases or remain symptomless. Several viroids have been reported to naturally infect pome and stone fruit trees, showing symptoms on leaves, fruits and/or bark. However, Koch&amp;rsquo;s postulates required for establishing on firm grounds the viroid etiology of these diseases, have not been met in all instances. Here, pome and stone fruit tree diseases, conclusively proven to be caused by viroids, are reviewed, and the need to pay closer attention to fulfilling Koch&amp;rsquo;s postulates is emphasized.

]]>Viroid Diseases in Pome and Stone Fruit Trees and Koch’s Postulates: A Critical AssessmentFrancesco Di SerioSilvia AmbrósTeruo SanoRicardo FloresBeatriz Navarrodoi: 10.3390/v10110612Viruses2018-11-07Viruses2018-11-071011Review61210.3390/v10110612http://www.mdpi.com/1999-4915/10/11/612Viruses, Vol. 10, Pages 611: Isolation of A Novel Bacillus thuringiensis Phage Representing A New Phage Lineage and Characterization of Its Endolysinhttp://www.mdpi.com/1999-4915/10/11/611
Phages, the parasites of bacteria, are considered as a new kind of antimicrobial agent due to their ability to lyse pathogenic bacteria. Due to the increase of available phage isolates, the newly isolated phage showed increasing genomic similarities with previously isolated phages. In this study, the novel phage vB_BthS_BMBphi, infecting the Bacillus thuringiensis strain BMB171, is isolated and characterized together with its endolysin. This phage is the first tadpole-like phage infecting the Bacillus strains. Genomic analysis shows that the phage genome is dissimilar to all those of previously characterized phages, only exhibiting low similarities with partial regions of the B. thuringiensis prophages. Phylogenetic analysis revealed that the phage was distant from the other Bacillus phages in terms of evolution. The novel genome sequence, the distant evolutionary relationship, and the special virion morphology together suggest that the phage vB_BthS_BMBphi could be classified as a new phage lineage. The genome of the phage is found to contain a restriction modification system, which might endow the phage with immunity to the restriction modification system of the host bacterium. The function of the endolysin PlyBMB encoded by the phage vB_BthS_BMBphi was analyzed, and the endolysin could lyse all the tested Bacillus cereus group strains, suggesting that the endolysin might be used in controlling pathogenic B. cereus group strains. The findings of this study enrich the understanding of phage diversity and provide a resource for controlling the B. cereus group pathogenic bacteria.Viruses, Vol. 10, Pages 611: Isolation of A Novel Bacillus thuringiensis Phage Representing A New Phage Lineage and Characterization of Its Endolysin

Phages, the parasites of bacteria, are considered as a new kind of antimicrobial agent due to their ability to lyse pathogenic bacteria. Due to the increase of available phage isolates, the newly isolated phage showed increasing genomic similarities with previously isolated phages. In this study, the novel phage vB_BthS_BMBphi, infecting the Bacillus thuringiensis strain BMB171, is isolated and characterized together with its endolysin. This phage is the first tadpole-like phage infecting the Bacillus strains. Genomic analysis shows that the phage genome is dissimilar to all those of previously characterized phages, only exhibiting low similarities with partial regions of the B. thuringiensis prophages. Phylogenetic analysis revealed that the phage was distant from the other Bacillus phages in terms of evolution. The novel genome sequence, the distant evolutionary relationship, and the special virion morphology together suggest that the phage vB_BthS_BMBphi could be classified as a new phage lineage. The genome of the phage is found to contain a restriction modification system, which might endow the phage with immunity to the restriction modification system of the host bacterium. The function of the endolysin PlyBMB encoded by the phage vB_BthS_BMBphi was analyzed, and the endolysin could lyse all the tested Bacillus cereus group strains, suggesting that the endolysin might be used in controlling pathogenic B. cereus group strains. The findings of this study enrich the understanding of phage diversity and provide a resource for controlling the B. cereus group pathogenic bacteria.

]]>Isolation of A Novel Bacillus thuringiensis Phage Representing A New Phage Lineage and Characterization of Its EndolysinYihui YuanQin PengShuo YangShaowen ZhangYajuan FuYan WuMeiying Gaodoi: 10.3390/v10110611Viruses2018-11-06Viruses2018-11-061011Article61110.3390/v10110611http://www.mdpi.com/1999-4915/10/11/611Viruses, Vol. 10, Pages 610: Combination Therapy with Oseltamivir and Favipiravir Delays Mortality but Does Not Prevent Oseltamivir Resistance in Immunodeficient Mice Infected with Pandemic A(H1N1) Influenza Virushttp://www.mdpi.com/1999-4915/10/11/610
Immunosuppressed individuals can shed influenza virus for prolonged periods of time, leading to the frequent emergence of antiviral resistance. We evaluated the benefits of oseltamivir and favipiravir combination therapy compared to single antiviral agents and monitored the emergence of drug-resistant variants in a pharmacologically immunosuppressed mouse model infected with the A(H1N1) pandemic influenza virus. C57BL/6 mice were immunosuppressed with cyclophosphamide and infected with a lethal dose of pandemic influenza A(H1N1) virus. Forty-eight hours post-infection, mice were treated with oseltamivir (20 mg/kg), favipiravir (20 or 50 mg/kg) or both agents BID for 5 or 10 days. Body weight losses, survival rates, lung viral titers, cytokine levels and emergence of resistant viruses were evaluated. Treatment of immunosuppressed mice with high (50 mg/kg) but not low (20 mg/kg) doses of favipiravir in combination with oseltamivir (20 mg/kg) significantly delayed mortality and reduced lung viral titers compared to treatment with a single drug regimen with oseltamivir but did not prevent the emergence of oseltamivir-resistant H275Y neuraminidase variants. Combination therapy with oseltamivir and favipiravir should be considered for evaluation in clinical trials.Viruses, Vol. 10, Pages 610: Combination Therapy with Oseltamivir and Favipiravir Delays Mortality but Does Not Prevent Oseltamivir Resistance in Immunodeficient Mice Infected with Pandemic A(H1N1) Influenza Virus

Immunosuppressed individuals can shed influenza virus for prolonged periods of time, leading to the frequent emergence of antiviral resistance. We evaluated the benefits of oseltamivir and favipiravir combination therapy compared to single antiviral agents and monitored the emergence of drug-resistant variants in a pharmacologically immunosuppressed mouse model infected with the A(H1N1) pandemic influenza virus. C57BL/6 mice were immunosuppressed with cyclophosphamide and infected with a lethal dose of pandemic influenza A(H1N1) virus. Forty-eight hours post-infection, mice were treated with oseltamivir (20 mg/kg), favipiravir (20 or 50 mg/kg) or both agents BID for 5 or 10 days. Body weight losses, survival rates, lung viral titers, cytokine levels and emergence of resistant viruses were evaluated. Treatment of immunosuppressed mice with high (50 mg/kg) but not low (20 mg/kg) doses of favipiravir in combination with oseltamivir (20 mg/kg) significantly delayed mortality and reduced lung viral titers compared to treatment with a single drug regimen with oseltamivir but did not prevent the emergence of oseltamivir-resistant H275Y neuraminidase variants. Combination therapy with oseltamivir and favipiravir should be considered for evaluation in clinical trials.

]]>Combination Therapy with Oseltamivir and Favipiravir Delays Mortality but Does Not Prevent Oseltamivir Resistance in Immunodeficient Mice Infected with Pandemic A(H1N1) Influenza VirusMariana BazJulie CarbonneauChantal RhéaumeMarie-Hélène CavanaghGuy Boivindoi: 10.3390/v10110610Viruses2018-11-03Viruses2018-11-031011Article61010.3390/v10110610http://www.mdpi.com/1999-4915/10/11/610Viruses, Vol. 10, Pages 609: Clinical Features of Varicella-Zoster Virus Infectionhttp://www.mdpi.com/1999-4915/10/11/609
Varicella-zoster virus (VZV) is a pathogenic human herpes virus that causes varicella (chickenpox) as a primary infection, following which it becomes latent in peripheral ganglia. Decades later, the virus may reactivate either spontaneously or after a number of triggering factors to cause herpes zoster (shingles). Varicella and its complications are more severe in the immunosuppressed. The most frequent and important complication of VZV reactivation is postherpetic neuralgia, the cause of which is unknown and for which treatment is usually ineffective. Reactivation of VZV may also cause a wide variety of neurological syndromes, the most significant of which is a vasculitis, which is treated with corticosteroids and the antiviral drug acyclovir. Other VZV reactivation complications include an encephalitis, segmental motor weakness and myelopathy, cranial neuropathies, Guillain&amp;ndash;Barr&amp;eacute; syndrome, enteric features, and zoster sine herpete, in which the viral reactivation occurs in the absence of the characteristic dermatomally distributed vesicular rash of herpes zoster. There has also been a recent association of VZV with giant cell arteritis and this interesting finding needs further corroboration. Vaccination is now available for the prevention of both varicella in children and herpes zoster in older individuals.Viruses, Vol. 10, Pages 609: Clinical Features of Varicella-Zoster Virus Infection

Varicella-zoster virus (VZV) is a pathogenic human herpes virus that causes varicella (chickenpox) as a primary infection, following which it becomes latent in peripheral ganglia. Decades later, the virus may reactivate either spontaneously or after a number of triggering factors to cause herpes zoster (shingles). Varicella and its complications are more severe in the immunosuppressed. The most frequent and important complication of VZV reactivation is postherpetic neuralgia, the cause of which is unknown and for which treatment is usually ineffective. Reactivation of VZV may also cause a wide variety of neurological syndromes, the most significant of which is a vasculitis, which is treated with corticosteroids and the antiviral drug acyclovir. Other VZV reactivation complications include an encephalitis, segmental motor weakness and myelopathy, cranial neuropathies, Guillain&amp;ndash;Barr&amp;eacute; syndrome, enteric features, and zoster sine herpete, in which the viral reactivation occurs in the absence of the characteristic dermatomally distributed vesicular rash of herpes zoster. There has also been a recent association of VZV with giant cell arteritis and this interesting finding needs further corroboration. Vaccination is now available for the prevention of both varicella in children and herpes zoster in older individuals.

]]>Clinical Features of Varicella-Zoster Virus InfectionPeter G. E. KennedyAnne A. Gershondoi: 10.3390/v10110609Viruses2018-11-02Viruses2018-11-021011Review60910.3390/v10110609http://www.mdpi.com/1999-4915/10/11/609Viruses, Vol. 10, Pages 608: High Thermal Diffusivity in Thermally Treated Filamentous Virus-Based Assemblies with a Smectic Liquid Crystalline Orientationhttp://www.mdpi.com/1999-4915/10/11/608
Polymers are generally considered thermal insulators because the amorphous arrangement of the polymeric chains reduces the mean free path of heat-conducting phonons. Recent studies reveal that individual chains of polymers with oriented structures could have high thermal conductivity, because such stretched polymeric chains effectively conduct phonons through polymeric covalent bonds. Previously, we have found that the liquid crystalline assembly composed of one of the filamentous viruses, M13 bacteriophages (M13 phages), shows high thermal diffusivity even though the assembly is based on non-covalent bonds. Despite such potential applicability of biopolymeric assemblies as thermal conductive materials, stability against heating has rarely been investigated. Herein, we demonstrate the maintenance of high thermal diffusivity in smectic liquid crystalline-oriented M13 phage-based assemblies after high temperature (150 &amp;deg;C) treatment. The liquid crystalline orientation of the M13 phage assemblies plays an important role in the stability against heating processes. Our results provide insight into the future use of biomolecular assemblies for reliable thermal conductive materials.Viruses, Vol. 10, Pages 608: High Thermal Diffusivity in Thermally Treated Filamentous Virus-Based Assemblies with a Smectic Liquid Crystalline Orientation

Polymers are generally considered thermal insulators because the amorphous arrangement of the polymeric chains reduces the mean free path of heat-conducting phonons. Recent studies reveal that individual chains of polymers with oriented structures could have high thermal conductivity, because such stretched polymeric chains effectively conduct phonons through polymeric covalent bonds. Previously, we have found that the liquid crystalline assembly composed of one of the filamentous viruses, M13 bacteriophages (M13 phages), shows high thermal diffusivity even though the assembly is based on non-covalent bonds. Despite such potential applicability of biopolymeric assemblies as thermal conductive materials, stability against heating has rarely been investigated. Herein, we demonstrate the maintenance of high thermal diffusivity in smectic liquid crystalline-oriented M13 phage-based assemblies after high temperature (150 &amp;deg;C) treatment. The liquid crystalline orientation of the M13 phage assemblies plays an important role in the stability against heating processes. Our results provide insight into the future use of biomolecular assemblies for reliable thermal conductive materials.

]]>High Thermal Diffusivity in Thermally Treated Filamentous Virus-Based Assemblies with a Smectic Liquid Crystalline OrientationToshiki SawadaYuta MurataHironori MarubayashiShuichi NojimaJunko MorikawaTakeshi Serizawadoi: 10.3390/v10110608Viruses2018-11-02Viruses2018-11-021011Article60810.3390/v10110608http://www.mdpi.com/1999-4915/10/11/608Viruses, Vol. 10, Pages 607: Transcriptomics Reveal Antiviral Gene Induction in the Egyptian Rousette Bat Is Antagonized In Vitro by Marburg Virus Infectionhttp://www.mdpi.com/1999-4915/10/11/607
The Egyptian rousette bat (ERB) is the only known Marburg virus (MARV) reservoir host. ERBs develop a productive MARV infection with low viremia and shedding but no overt disease, suggesting this virus is efficiently controlled by ERB antiviral responses. This dynamic would contrast with humans, where MARV-mediated interferon (IFN) antagonism early in infection is thought to contribute to the severe, often fatal disease. The newly-annotated ERB genome and transcriptome have now enabled us to use a custom-designed NanoString nCounter ERB CodeSet in conjunction with RNA-seq to investigate responses in a MARV-infected ERB cell line. Both transcriptomic platforms correlated well and showed that MARV inhibited the antiviral program in ERB cells, while an IFN antagonism-impaired MARV was less efficient at suppressing the response gene induction, phenotypes previously reported for primate cells. Interestingly, and despite the expansion of IFN loci in the ERB genome, neither MARV showed specific induction of almost any IFN gene. However, we detected an upregulation of putative, unannotated ERB antiviral paralogs, as well as an elevated basal expression in uninfected ERB cells of key antiviral genes.Viruses, Vol. 10, Pages 607: Transcriptomics Reveal Antiviral Gene Induction in the Egyptian Rousette Bat Is Antagonized In Vitro by Marburg Virus Infection

The Egyptian rousette bat (ERB) is the only known Marburg virus (MARV) reservoir host. ERBs develop a productive MARV infection with low viremia and shedding but no overt disease, suggesting this virus is efficiently controlled by ERB antiviral responses. This dynamic would contrast with humans, where MARV-mediated interferon (IFN) antagonism early in infection is thought to contribute to the severe, often fatal disease. The newly-annotated ERB genome and transcriptome have now enabled us to use a custom-designed NanoString nCounter ERB CodeSet in conjunction with RNA-seq to investigate responses in a MARV-infected ERB cell line. Both transcriptomic platforms correlated well and showed that MARV inhibited the antiviral program in ERB cells, while an IFN antagonism-impaired MARV was less efficient at suppressing the response gene induction, phenotypes previously reported for primate cells. Interestingly, and despite the expansion of IFN loci in the ERB genome, neither MARV showed specific induction of almost any IFN gene. However, we detected an upregulation of putative, unannotated ERB antiviral paralogs, as well as an elevated basal expression in uninfected ERB cells of key antiviral genes.

]]>Transcriptomics Reveal Antiviral Gene Induction in the Egyptian Rousette Bat Is Antagonized In Vitro by Marburg Virus InfectionCatherine E. ArnoldJonathan C. GuitoLouis A. AltamuraSean P. LovettElyse R. NagleGustavo F. PalaciosMariano Sanchez-LockhartJonathan S. Townerdoi: 10.3390/v10110607Viruses2018-11-02Viruses2018-11-021011Article60710.3390/v10110607http://www.mdpi.com/1999-4915/10/11/607Viruses, Vol. 10, Pages 606: Mitochondrial-Directed Antioxidant Reduces Microglial-Induced Inflammation in Murine In Vitro Model of TC-83 Infectionhttp://www.mdpi.com/1999-4915/10/11/606
Venezuelan equine encephalitis virus (VEEV) is an arbovirus that is associated with robust inflammation that contributes to neurodegenerative phenotypes. In addition to triggering central nervous system (CNS) inflammation, VEEV will also induce mitochondrial dysfunction, resulting in increased cellular apoptosis. In this study, we utilize the TC-83 strain of VEEV to determine the role of mitochondrial oxidative stress in mediating inflammation elicited by murine brain microglial cells. Using an in vitro model, we show that murine microglia are susceptible to TC-83 infection, and that these cells undergo mitochondrial stress as the result of infection. We also indicate that bystander microglia contribute more significantly to the overall inflammatory load than directly infected microglia. Use of a mitochondrial targeted antioxidant, mitoquinone mesylate, greatly reduced the pro-inflammatory cytokines released by both direct infected and bystander microglia. Our data suggest that release of interleukin-1&amp;beta;, a key instigator of neuroinflammation during VEEV infection, may be the direct result of accumulating mitochondrial stress. This data improves our understanding inflammation elicited by murine microglia and will aid in the development of more accurate in vitro and in vivo murine model of VEEV-induced neuroinflammation.Viruses, Vol. 10, Pages 606: Mitochondrial-Directed Antioxidant Reduces Microglial-Induced Inflammation in Murine In Vitro Model of TC-83 Infection

Venezuelan equine encephalitis virus (VEEV) is an arbovirus that is associated with robust inflammation that contributes to neurodegenerative phenotypes. In addition to triggering central nervous system (CNS) inflammation, VEEV will also induce mitochondrial dysfunction, resulting in increased cellular apoptosis. In this study, we utilize the TC-83 strain of VEEV to determine the role of mitochondrial oxidative stress in mediating inflammation elicited by murine brain microglial cells. Using an in vitro model, we show that murine microglia are susceptible to TC-83 infection, and that these cells undergo mitochondrial stress as the result of infection. We also indicate that bystander microglia contribute more significantly to the overall inflammatory load than directly infected microglia. Use of a mitochondrial targeted antioxidant, mitoquinone mesylate, greatly reduced the pro-inflammatory cytokines released by both direct infected and bystander microglia. Our data suggest that release of interleukin-1&amp;beta;, a key instigator of neuroinflammation during VEEV infection, may be the direct result of accumulating mitochondrial stress. This data improves our understanding inflammation elicited by murine microglia and will aid in the development of more accurate in vitro and in vivo murine model of VEEV-induced neuroinflammation.

]]>Mitochondrial-Directed Antioxidant Reduces Microglial-Induced Inflammation in Murine In Vitro Model of TC-83 InfectionForrest KeckDaud KhanBrian RobertsNitin AgrawalNishank BhallaAarthi Narayanandoi: 10.3390/v10110606Viruses2018-11-02Viruses2018-11-021011Article60610.3390/v10110606http://www.mdpi.com/1999-4915/10/11/606Viruses, Vol. 10, Pages 605: Genetic Resistance to Avian Leukosis Viruses Induced by CRISPR/Cas9 Editing of Specific Receptor Genes in Chicken Cellshttp://www.mdpi.com/1999-4915/10/11/605
Avian leukosis viruses (ALVs), which are pathogens of concern in domestic poultry, utilize specific receptor proteins for cell entry that are both necessary and sufficient for host susceptibility to a given ALV subgroup. This unequivocal relationship offers receptors as suitable targets of selection and biotechnological manipulation with the aim of obtaining virus-resistant poultry. This approach is further supported by the existence of natural knock-outs of receptor genes that segregate in inbred lines of chickens. We used CRISPR/Cas9 genome editing tools to introduce frame-shifting indel mutations into tva, tvc, and tvj loci encoding receptors for the A, C, and J ALV subgroups, respectively. For all three loci, the homozygous frame-shifting indels generating premature stop codons induced phenotypes which were fully resistant to the virus of respective subgroup. In the tvj locus, we also obtained in-frame deletions corroborating the importance of W38 and the four amino-acids preceding it. We demonstrate that CRISPR/Cas9-mediated knock-out or the fine editing of ALV receptor genes might be the first step in the development of virus-resistant chickens.Viruses, Vol. 10, Pages 605: Genetic Resistance to Avian Leukosis Viruses Induced by CRISPR/Cas9 Editing of Specific Receptor Genes in Chicken Cells

Avian leukosis viruses (ALVs), which are pathogens of concern in domestic poultry, utilize specific receptor proteins for cell entry that are both necessary and sufficient for host susceptibility to a given ALV subgroup. This unequivocal relationship offers receptors as suitable targets of selection and biotechnological manipulation with the aim of obtaining virus-resistant poultry. This approach is further supported by the existence of natural knock-outs of receptor genes that segregate in inbred lines of chickens. We used CRISPR/Cas9 genome editing tools to introduce frame-shifting indel mutations into tva, tvc, and tvj loci encoding receptors for the A, C, and J ALV subgroups, respectively. For all three loci, the homozygous frame-shifting indels generating premature stop codons induced phenotypes which were fully resistant to the virus of respective subgroup. In the tvj locus, we also obtained in-frame deletions corroborating the importance of W38 and the four amino-acids preceding it. We demonstrate that CRISPR/Cas9-mediated knock-out or the fine editing of ALV receptor genes might be the first step in the development of virus-resistant chickens.

]]>Genetic Resistance to Avian Leukosis Viruses Induced by CRISPR/Cas9 Editing of Specific Receptor Genes in Chicken CellsAnna KoslováDana KučerováMarkéta ReinišováJosef GerykPavel TrefilJiří Hejnardoi: 10.3390/v10110605Viruses2018-11-02Viruses2018-11-021011Article60510.3390/v10110605http://www.mdpi.com/1999-4915/10/11/605Viruses, Vol. 10, Pages 604: Evolution of Codon Usage Bias in Henipaviruses Is Governed by Natural Selection and Is Host-Specifichttp://www.mdpi.com/1999-4915/10/11/604
Hendra virus (HeV) and Nipah virus (NiV) are among a group of emerging bat-borne paramyxoviruses that have crossed their species-barrier several times by infecting several hosts with a high fatality rate in human beings. Despite the fatal nature of their infection, a comprehensive study to explore their evolution and adaptation in different hosts is lacking. A study of codon usage patterns in henipaviruses may provide some fruitful insight into their evolutionary processes of synonymous codon usage and host-adapted evolution. Here, we performed a systematic evolutionary and codon usage bias analysis of henipaviruses. We found a low codon usage bias in the coding sequences of henipaviruses and that natural selection, mutation pressure, and nucleotide compositions shapes the codon usage patterns of henipaviruses, with natural selection being more important than the others. Also, henipaviruses showed the highest level of adaptation to bats of the genus Pteropus in the codon adaptation index (CAI), relative to the codon de-optimization index (RCDI), and similarity index (SiD) analyses. Furthermore, a comparison to recently identified henipa-like viruses indicated a high tRNA adaptation index of henipaviruses for human beings, mainly due to F, G and L proteins. Consequently, the study concedes the substantial emergence of henipaviruses in human beings, particularly when paired with frequent exposure to direct/indirect bat excretions.Viruses, Vol. 10, Pages 604: Evolution of Codon Usage Bias in Henipaviruses Is Governed by Natural Selection and Is Host-Specific

Hendra virus (HeV) and Nipah virus (NiV) are among a group of emerging bat-borne paramyxoviruses that have crossed their species-barrier several times by infecting several hosts with a high fatality rate in human beings. Despite the fatal nature of their infection, a comprehensive study to explore their evolution and adaptation in different hosts is lacking. A study of codon usage patterns in henipaviruses may provide some fruitful insight into their evolutionary processes of synonymous codon usage and host-adapted evolution. Here, we performed a systematic evolutionary and codon usage bias analysis of henipaviruses. We found a low codon usage bias in the coding sequences of henipaviruses and that natural selection, mutation pressure, and nucleotide compositions shapes the codon usage patterns of henipaviruses, with natural selection being more important than the others. Also, henipaviruses showed the highest level of adaptation to bats of the genus Pteropus in the codon adaptation index (CAI), relative to the codon de-optimization index (RCDI), and similarity index (SiD) analyses. Furthermore, a comparison to recently identified henipa-like viruses indicated a high tRNA adaptation index of henipaviruses for human beings, mainly due to F, G and L proteins. Consequently, the study concedes the substantial emergence of henipaviruses in human beings, particularly when paired with frequent exposure to direct/indirect bat excretions.

]]>Evolution of Codon Usage Bias in Henipaviruses Is Governed by Natural Selection and Is Host-SpecificNaveen KumarDiwakar D. KulkarniBenhur LeeRahul KaushikSandeep BhatiaRicha SoodAtul Kumar PateriyaSushant BhatVijendra Pal Singhdoi: 10.3390/v10110604Viruses2018-11-01Viruses2018-11-011011Article60410.3390/v10110604http://www.mdpi.com/1999-4915/10/11/604Viruses, Vol. 10, Pages 603: Comprehensive Analysis of Hepatitis B Virus Promoter Region Mutationshttp://www.mdpi.com/1999-4915/10/11/603
Over 250 million people are infected chronically with hepatitis B virus (HBV), the leading cause of liver cancer worldwide. HBV persists, due, in part, to its compact, stable minichromosome, the covalently-closed, circular DNA (cccDNA), which resides in the hepatocytes&amp;rsquo; nuclei. Current therapies target downstream replication products, however, a true virological cure will require targeting the cccDNA. Finding targets on such a small, compact genome is challenging. For HBV, to remain replication-competent, it needs to maintain nucleotide fidelity in key regions, such as the promoter regions, to ensure that it can continue to utilize the necessary host proteins. HBVdb (HBV database) is a repository of HBV sequences spanning all genotypes (A&amp;ndash;H) amplified from clinical samples, and hence implying an extensive collection of replication-competent viruses. Here, we analyzed the HBV sequences from HBVdb using bioinformatics tools to comprehensively assess the HBV core and X promoter regions amongst the nearly 70,000 HBV sequences for highly-conserved nucleotides and variant frequencies. Notably, there is a high degree of nucleotide conservation within specific segments of these promoter regions highlighting their importance in potential host protein-viral interactions and thus the virus&amp;rsquo; viability. Such findings may have key implications for designing antivirals to target these areas.Viruses, Vol. 10, Pages 603: Comprehensive Analysis of Hepatitis B Virus Promoter Region Mutations

Over 250 million people are infected chronically with hepatitis B virus (HBV), the leading cause of liver cancer worldwide. HBV persists, due, in part, to its compact, stable minichromosome, the covalently-closed, circular DNA (cccDNA), which resides in the hepatocytes&amp;rsquo; nuclei. Current therapies target downstream replication products, however, a true virological cure will require targeting the cccDNA. Finding targets on such a small, compact genome is challenging. For HBV, to remain replication-competent, it needs to maintain nucleotide fidelity in key regions, such as the promoter regions, to ensure that it can continue to utilize the necessary host proteins. HBVdb (HBV database) is a repository of HBV sequences spanning all genotypes (A&amp;ndash;H) amplified from clinical samples, and hence implying an extensive collection of replication-competent viruses. Here, we analyzed the HBV sequences from HBVdb using bioinformatics tools to comprehensively assess the HBV core and X promoter regions amongst the nearly 70,000 HBV sequences for highly-conserved nucleotides and variant frequencies. Notably, there is a high degree of nucleotide conservation within specific segments of these promoter regions highlighting their importance in potential host protein-viral interactions and thus the virus&amp;rsquo; viability. Such findings may have key implications for designing antivirals to target these areas.

]]>Comprehensive Analysis of Hepatitis B Virus Promoter Region MutationsVanessa Meier-StephensonWilliam T. R. BremnerChimone S. DaltonGuido van MarleCarla S. CoffinTrushar R. Pateldoi: 10.3390/v10110603Viruses2018-11-01Viruses2018-11-011011Article60310.3390/v10110603http://www.mdpi.com/1999-4915/10/11/603Viruses, Vol. 10, Pages 602: CRISPRStudio: A User-Friendly Software for Rapid CRISPR Array Visualizationhttp://www.mdpi.com/1999-4915/10/11/602
The CRISPR-Cas system biologically serves as an adaptive defense mechanism against phages. However, there is growing interest in exploiting the hypervariable nature of the CRISPR locus, often of viral origin, for microbial typing and tracking. Moreover, the spacer content of any given strain provides a phage resistance profile. Large-scale CRISPR typing studies require an efficient method for showcasing CRISPR array similarities across multiple isolates. Historically, CRISPR arrays found in microbes have been represented by colored shapes based on nucleotide sequence identity and, while this approach is now routinely used, only scarce computational resources are available to automate the process, making it very time-consuming for large datasets. To alleviate this tedious task, we introduce CRISPRStudio, a command-line tool developed to accelerate CRISPR analysis and standardize the preparation of CRISPR array figures. It first compares nucleotide spacer sequences present in a dataset and then clusters them based on sequence similarity to assign a meaningful representative color. CRISPRStudio offers versatility to suit different biological contexts by including options such as automatic sorting of CRISPR loci and highlighting of shared spacers, while remaining fast and user-friendly.Viruses, Vol. 10, Pages 602: CRISPRStudio: A User-Friendly Software for Rapid CRISPR Array Visualization

The CRISPR-Cas system biologically serves as an adaptive defense mechanism against phages. However, there is growing interest in exploiting the hypervariable nature of the CRISPR locus, often of viral origin, for microbial typing and tracking. Moreover, the spacer content of any given strain provides a phage resistance profile. Large-scale CRISPR typing studies require an efficient method for showcasing CRISPR array similarities across multiple isolates. Historically, CRISPR arrays found in microbes have been represented by colored shapes based on nucleotide sequence identity and, while this approach is now routinely used, only scarce computational resources are available to automate the process, making it very time-consuming for large datasets. To alleviate this tedious task, we introduce CRISPRStudio, a command-line tool developed to accelerate CRISPR analysis and standardize the preparation of CRISPR array figures. It first compares nucleotide spacer sequences present in a dataset and then clusters them based on sequence similarity to assign a meaningful representative color. CRISPRStudio offers versatility to suit different biological contexts by including options such as automatic sorting of CRISPR loci and highlighting of shared spacers, while remaining fast and user-friendly.

To complement traditional antivirals, natural compounds that act via host targets and present high barriers to resistance are of increasing interest. In the work reported here, we detected that homoharringtonine (HHT) presents effective antiviral activity. HHT completely inhibited infections of vesicular stomatitis virus (VSV), Newcastle disease virus (NDV), and porcine epidemic diarrhea virus (PEDV) at concentrations of 50, 100, and 500 nM in cell cultures, respectively. Treatment with HHT at doses of 0.05 or 0.2 mg/kg significantly reduced viral load and relieved severe symptoms in PEDV- or NDV-infected animals. HHT treatment, however, moderately inhibited avian influenza virus (AIV) infection, suggesting its potent antiviral action is restricted to a number of classes of RNA viruses. In this study, we also observed that HHT actively inhibited herpes simplex virus type 1 (HSV-1) replication with a 50% inhibitory concentration (IC50) of 139 nM; the treatment with HHT at 1000 nM led to reductions of three orders of magnitude. Moreover, HHT antagonized the phosphorylation level of endogenous and exogenous eukaryotic initiation factor 4E (p-eIF4E), which might regulate the selective translation of specific messenger RNA (mRNA). HHT provides a starting point for further progress toward the clinical development of broad-spectrum antivirals.

]]>The Natural Compound Homoharringtonine Presents Broad Antiviral Activity In Vitro and In VivoHui-Jun DongZhao-Hua WangWen MengCui-Cui LiYan-Xin HuLei ZhouXiao-Jia Wangdoi: 10.3390/v10110601Viruses2018-11-01Viruses2018-11-011011Article60110.3390/v10110601http://www.mdpi.com/1999-4915/10/11/601Viruses, Vol. 10, Pages 600: RNA Virus Fidelity Mutants: A Useful Tool for Evolutionary Biology or a Complex Challenge?http://www.mdpi.com/1999-4915/10/11/600
RNA viruses replicate with low fidelity due to the error-prone nature of the RNA-dependent RNA polymerase, which generates approximately one mutation per round of genome replication. Due to the large population sizes produced by RNA viruses during replication, this results in a cloud of closely related virus variants during host infection, of which small increases or decreases in replication fidelity have been shown to result in virus attenuation in vivo, but not typically in vitro. Since the discovery of the first RNA virus fidelity mutants during the mid-aughts, the field has exploded with the identification of over 50 virus fidelity mutants distributed amongst 7 RNA virus families. This review summarizes the current RNA virus fidelity mutant literature, with a focus upon the definition of a fidelity mutant as well as methods to confirm any mutational changes associated with the fidelity mutant. Due to the complexity of such a definition, in addition to reports of unstable virus fidelity phenotypes, the future translational utility of these mutants and applications for basic science are examined.Viruses, Vol. 10, Pages 600: RNA Virus Fidelity Mutants: A Useful Tool for Evolutionary Biology or a Complex Challenge?

RNA viruses replicate with low fidelity due to the error-prone nature of the RNA-dependent RNA polymerase, which generates approximately one mutation per round of genome replication. Due to the large population sizes produced by RNA viruses during replication, this results in a cloud of closely related virus variants during host infection, of which small increases or decreases in replication fidelity have been shown to result in virus attenuation in vivo, but not typically in vitro. Since the discovery of the first RNA virus fidelity mutants during the mid-aughts, the field has exploded with the identification of over 50 virus fidelity mutants distributed amongst 7 RNA virus families. This review summarizes the current RNA virus fidelity mutant literature, with a focus upon the definition of a fidelity mutant as well as methods to confirm any mutational changes associated with the fidelity mutant. Due to the complexity of such a definition, in addition to reports of unstable virus fidelity phenotypes, the future translational utility of these mutants and applications for basic science are examined.

]]>RNA Virus Fidelity Mutants: A Useful Tool for Evolutionary Biology or a Complex Challenge?Tiffany F. KautzNaomi L. Forresterdoi: 10.3390/v10110600Viruses2018-11-01Viruses2018-11-011011Review60010.3390/v10110600http://www.mdpi.com/1999-4915/10/11/600Viruses, Vol. 10, Pages 599: EBV and KSHV Infection Dysregulates Autophagy to Optimize Viral Replication, Prevent Immune Recognition and Promote Tumorigenesishttp://www.mdpi.com/1999-4915/10/11/599
Autophagy is a catabolic process strongly involved in the immune response, and its dysregulation contributes to the onset of several diseases including cancer. The human oncogenic gammaherpesviruses, Epstein&amp;mdash;Barr virus (EBV) and Kaposi&amp;rsquo;s sarcoma-associated herpesvirus (KSHV), manipulate autophagy, either during the de novo infection or during the lytic reactivation, in naturally latently-infected lymphoma cells. In particular, the gammaherpesvirus infection reduces autophagy in immune cells, such as monocytes, resulting in the impairment of cell survival and cell differentiation into dendritic cells (DCs), which are essential for initiating and regulating the immune response. In the case of EBV, the reduction of autophagy in these cells, leading to p62 accumulation, activated the p62-NRF2-antioxidant response, reducing ROS, and further inhibiting autophagy. KSHV inhibits autophagy in monocytes by de-phosphorylating JNK2, altering the calpains&amp;ndash;calpastatin balance and increasing the calpain activity responsible for the cleavage of ATG5. To further impair the immune response, KSHV also inhibits autophagy in differentiated DCs by hyper-phosphorylating STAT3. Conversely, when the lytic cycle is induced in vitro in latently-infected lymphoma B cells, both EBV and KSHV promote autophagy to enhance their replication, although the final autophagic steps are blocked through the down-regulation of Rab7. This strategy allows viruses to avoid the destructive environment of lysosomes, and to exploit the autophagic machinery for intracellular transportation. EBV and KSHV encode for proteins that may either inhibit or promote autophagy and, in addition, they can modulate the cellular pathways that control this process. In this review we will discuss the findings that indicate that autophagy is dysregulated by gammaherpesvirus to promote immune suppression, facilitate viral replication and contribute to the onset and maintenance of gammaherpesvirus-associated malignancies.Viruses, Vol. 10, Pages 599: EBV and KSHV Infection Dysregulates Autophagy to Optimize Viral Replication, Prevent Immune Recognition and Promote Tumorigenesis

Autophagy is a catabolic process strongly involved in the immune response, and its dysregulation contributes to the onset of several diseases including cancer. The human oncogenic gammaherpesviruses, Epstein&amp;mdash;Barr virus (EBV) and Kaposi&amp;rsquo;s sarcoma-associated herpesvirus (KSHV), manipulate autophagy, either during the de novo infection or during the lytic reactivation, in naturally latently-infected lymphoma cells. In particular, the gammaherpesvirus infection reduces autophagy in immune cells, such as monocytes, resulting in the impairment of cell survival and cell differentiation into dendritic cells (DCs), which are essential for initiating and regulating the immune response. In the case of EBV, the reduction of autophagy in these cells, leading to p62 accumulation, activated the p62-NRF2-antioxidant response, reducing ROS, and further inhibiting autophagy. KSHV inhibits autophagy in monocytes by de-phosphorylating JNK2, altering the calpains&amp;ndash;calpastatin balance and increasing the calpain activity responsible for the cleavage of ATG5. To further impair the immune response, KSHV also inhibits autophagy in differentiated DCs by hyper-phosphorylating STAT3. Conversely, when the lytic cycle is induced in vitro in latently-infected lymphoma B cells, both EBV and KSHV promote autophagy to enhance their replication, although the final autophagic steps are blocked through the down-regulation of Rab7. This strategy allows viruses to avoid the destructive environment of lysosomes, and to exploit the autophagic machinery for intracellular transportation. EBV and KSHV encode for proteins that may either inhibit or promote autophagy and, in addition, they can modulate the cellular pathways that control this process. In this review we will discuss the findings that indicate that autophagy is dysregulated by gammaherpesvirus to promote immune suppression, facilitate viral replication and contribute to the onset and maintenance of gammaherpesvirus-associated malignancies.

]]>EBV and KSHV Infection Dysregulates Autophagy to Optimize Viral Replication, Prevent Immune Recognition and Promote TumorigenesisMara Cironedoi: 10.3390/v10110599Viruses2018-10-31Viruses2018-10-311011Review59910.3390/v10110599http://www.mdpi.com/1999-4915/10/11/599Viruses, Vol. 10, Pages 598: Animal Models of Zika Virus Infection during Pregnancyhttp://www.mdpi.com/1999-4915/10/11/598
Zika virus (ZIKV) emerged suddenly in the Americas in 2015 and was associated with a widespread outbreak of microcephaly and other severe congenital abnormalities in infants born to mothers infected during pregnancy. Vertical transmission of ZIKV in humans was confirmed when viral RNA was detected in fetal and placental tissues, and this outcome has been recapitulated experimentally in animals. Unlike other flaviviruses, ZIKV is both arthropod- and sexually-transmitted, and has a broad tissue tropism in humans, including multiple tissues of the reproductive tract. The threats posed by ZIKV have prompted the development of multiple in vivo models to better understand the pathogenesis of ZIKV, particularly during pregnancy. Here, we review the progress on animal models of ZIKV infection during pregnancy. These studies have generated a foundation of insights into the biology of ZIKV, and provide a means for evaluating vaccines and therapeutics.Viruses, Vol. 10, Pages 598: Animal Models of Zika Virus Infection during Pregnancy

Zika virus (ZIKV) emerged suddenly in the Americas in 2015 and was associated with a widespread outbreak of microcephaly and other severe congenital abnormalities in infants born to mothers infected during pregnancy. Vertical transmission of ZIKV in humans was confirmed when viral RNA was detected in fetal and placental tissues, and this outcome has been recapitulated experimentally in animals. Unlike other flaviviruses, ZIKV is both arthropod- and sexually-transmitted, and has a broad tissue tropism in humans, including multiple tissues of the reproductive tract. The threats posed by ZIKV have prompted the development of multiple in vivo models to better understand the pathogenesis of ZIKV, particularly during pregnancy. Here, we review the progress on animal models of ZIKV infection during pregnancy. These studies have generated a foundation of insights into the biology of ZIKV, and provide a means for evaluating vaccines and therapeutics.

]]>Animal Models of Zika Virus Infection during PregnancyElizabeth A. CaineBrett W. JaggerMichael S. Diamonddoi: 10.3390/v10110598Viruses2018-10-31Viruses2018-10-311011Review59810.3390/v10110598http://www.mdpi.com/1999-4915/10/11/598Viruses, Vol. 10, Pages 597: Reverse Genetic Approaches for the Generation of Recombinant Zika Virushttp://www.mdpi.com/1999-4915/10/11/597
Zika virus (ZIKV) is an emergent mosquito-borne member of the Flaviviridae family that was responsible for a recent epidemic in the Americas. ZIKV has been associated with severe clinical complications, including neurological disorder such as Guillain-Barr&amp;eacute; syndrome in adults and severe fetal abnormalities and microcephaly in newborn infants. Given the significance of these clinical manifestations, the development of tools and reagents to study the pathogenesis of ZIKV and to develop new therapeutic options are urgently needed. In this respect, the implementation of reverse genetic techniques has allowed the direct manipulation of the viral genome to generate recombinant (r)ZIKVs, which have provided investigators with powerful systems to answer important questions about the biology of ZIKV, including virus-host interactions, the mechanism of transmission and pathogenesis or the function of viral proteins. In this review, we will summarize the different reverse genetic strategies that have been implemented, to date, for the generation of rZIKVs and the applications of these platforms for the development of replicon systems or reporter-expressing viruses.Viruses, Vol. 10, Pages 597: Reverse Genetic Approaches for the Generation of Recombinant Zika Virus

Zika virus (ZIKV) is an emergent mosquito-borne member of the Flaviviridae family that was responsible for a recent epidemic in the Americas. ZIKV has been associated with severe clinical complications, including neurological disorder such as Guillain-Barr&amp;eacute; syndrome in adults and severe fetal abnormalities and microcephaly in newborn infants. Given the significance of these clinical manifestations, the development of tools and reagents to study the pathogenesis of ZIKV and to develop new therapeutic options are urgently needed. In this respect, the implementation of reverse genetic techniques has allowed the direct manipulation of the viral genome to generate recombinant (r)ZIKVs, which have provided investigators with powerful systems to answer important questions about the biology of ZIKV, including virus-host interactions, the mechanism of transmission and pathogenesis or the function of viral proteins. In this review, we will summarize the different reverse genetic strategies that have been implemented, to date, for the generation of rZIKVs and the applications of these platforms for the development of replicon systems or reporter-expressing viruses.

]]>Reverse Genetic Approaches for the Generation of Recombinant Zika VirusGinés Ávila-PérezAitor NogalesVerónica MartínFernando AlmazánLuis Martínez-Sobridodoi: 10.3390/v10110597Viruses2018-10-31Viruses2018-10-311011Review59710.3390/v10110597http://www.mdpi.com/1999-4915/10/11/597Viruses, Vol. 10, Pages 596: Immunogenicity of Pigeon Circovirus Recombinant Capsid Protein in Pigeonshttp://www.mdpi.com/1999-4915/10/11/596
Pigeon circovirus (PiCV) is the most frequently diagnosed virus in pigeons and is thought to be one of the causative factors of a complex disease called the young pigeon disease syndrome (YPDS). The development of a vaccine against this virus could be a strategy for YPDS control. Since laboratory culture of PiCV is impossible, its recombinant capsid protein (rCP) can be considered as a potential antigen candidate in sub-unit vaccines. The aim of this basic research was to evaluate the immune response of pigeons to PiCV rCP. Sixty six-week-old carrier pigeons were divided into two groups (experimental immunized with PiCV rCP mixed with an adjuvant, and control immunized with an adjuvant only), and immunized twice in a 21-day interval. On the day of immunization and on two, 23, 39, and 46 days post first immunization (dpv), samples of blood, spleen, and bursa of Fabricius were collected from six birds from each group to examine anti-PiCV rCP IgY, anti-PiCV rCP IgY-secreting B cells (SBC), IFN-&amp;gamma; gene expression, and percentage of T CD3+, CD4+, CD8+, and B IgM+ lymphocytes. The results indicated a correct immune response to PiCV rCP both in humoral and cell-mediated immunity, which was manifested by seroconversion since 23 dpv, by a significantly higher anti-PiCV rCP IgY-SBC number on two and 23 dpv, and significantly higher IFN-&amp;gamma; gene expression since two dpv. There were no significant differences or trends noted between particular T and B lymphocyte subpopulations. To conclude, PiCV rCP may be deemed immunogenic and could be considered as an antigen candidate in sub-unit vaccines against PiCV infections in pigeons.Viruses, Vol. 10, Pages 596: Immunogenicity of Pigeon Circovirus Recombinant Capsid Protein in Pigeons

Pigeon circovirus (PiCV) is the most frequently diagnosed virus in pigeons and is thought to be one of the causative factors of a complex disease called the young pigeon disease syndrome (YPDS). The development of a vaccine against this virus could be a strategy for YPDS control. Since laboratory culture of PiCV is impossible, its recombinant capsid protein (rCP) can be considered as a potential antigen candidate in sub-unit vaccines. The aim of this basic research was to evaluate the immune response of pigeons to PiCV rCP. Sixty six-week-old carrier pigeons were divided into two groups (experimental immunized with PiCV rCP mixed with an adjuvant, and control immunized with an adjuvant only), and immunized twice in a 21-day interval. On the day of immunization and on two, 23, 39, and 46 days post first immunization (dpv), samples of blood, spleen, and bursa of Fabricius were collected from six birds from each group to examine anti-PiCV rCP IgY, anti-PiCV rCP IgY-secreting B cells (SBC), IFN-&amp;gamma; gene expression, and percentage of T CD3+, CD4+, CD8+, and B IgM+ lymphocytes. The results indicated a correct immune response to PiCV rCP both in humoral and cell-mediated immunity, which was manifested by seroconversion since 23 dpv, by a significantly higher anti-PiCV rCP IgY-SBC number on two and 23 dpv, and significantly higher IFN-&amp;gamma; gene expression since two dpv. There were no significant differences or trends noted between particular T and B lymphocyte subpopulations. To conclude, PiCV rCP may be deemed immunogenic and could be considered as an antigen candidate in sub-unit vaccines against PiCV infections in pigeons.

]]>Immunogenicity of Pigeon Circovirus Recombinant Capsid Protein in PigeonsTomasz StenzelDaria DziewulskaBartłomiej TykałowskiMarcin ŚmiałekJoanna KowalczykAndrzej Koncickidoi: 10.3390/v10110596Viruses2018-10-31Viruses2018-10-311011Article59610.3390/v10110596http://www.mdpi.com/1999-4915/10/11/596Viruses, Vol. 10, Pages 595: Comparative Genomics and Characterization of the Late Promoter pR’ from Shiga Toxin Prophages in Escherichia colihttp://www.mdpi.com/1999-4915/10/11/595
Shiga-toxin producing Escherichia coli (STEC) causes human illness ranging from mild diarrhea to death. The bacteriophage encoded stx genes are located in the late transcription region, downstream of the antiterminator Q. The transcription of the stx genes is directly under the control of the late promoter pR&amp;rsquo;, thus the sequence diversity of the region between Q and stx, here termed the pR&amp;rsquo; region, may affect Stx toxin production. Here, we compared the gene structure of the pR&amp;rsquo; region and the stx subtypes of nineteen STECs. The sequence alignment and phylogenetic analysis suggested that the pR&amp;rsquo; region tends to be more heterogeneous than the promoter itself, even if the prophages harbor the same stx subtype. Furthermore, we established and validated transcriptional fusions of the pR&amp;rsquo; region to the DsRed reporter gene using mitomycin C (MMC) induction. Finally, these constructs were transformed into native and non-native strains and examined with flow cytometry. The results showed that induction levels changed when pR&amp;rsquo; regions were placed under different regulatory systems. Moreover, not every stx gene could be induced in its native host bacteria. In addition to the functional genes, the diversity of the pR&amp;rsquo; region plays an important role in determining the level of toxin induction.Viruses, Vol. 10, Pages 595: Comparative Genomics and Characterization of the Late Promoter pR’ from Shiga Toxin Prophages in Escherichia coli

Shiga-toxin producing Escherichia coli (STEC) causes human illness ranging from mild diarrhea to death. The bacteriophage encoded stx genes are located in the late transcription region, downstream of the antiterminator Q. The transcription of the stx genes is directly under the control of the late promoter pR&amp;rsquo;, thus the sequence diversity of the region between Q and stx, here termed the pR&amp;rsquo; region, may affect Stx toxin production. Here, we compared the gene structure of the pR&amp;rsquo; region and the stx subtypes of nineteen STECs. The sequence alignment and phylogenetic analysis suggested that the pR&amp;rsquo; region tends to be more heterogeneous than the promoter itself, even if the prophages harbor the same stx subtype. Furthermore, we established and validated transcriptional fusions of the pR&amp;rsquo; region to the DsRed reporter gene using mitomycin C (MMC) induction. Finally, these constructs were transformed into native and non-native strains and examined with flow cytometry. The results showed that induction levels changed when pR&amp;rsquo; regions were placed under different regulatory systems. Moreover, not every stx gene could be induced in its native host bacteria. In addition to the functional genes, the diversity of the pR&amp;rsquo; region plays an important role in determining the level of toxin induction.

]]>Comparative Genomics and Characterization of the Late Promoter pR’ from Shiga Toxin Prophages in Escherichia coliLing Xiao ZhangDavid J. SimpsonLynn M. McMullenMichael G. Gänzledoi: 10.3390/v10110595Viruses2018-10-31Viruses2018-10-311011Article59510.3390/v10110595http://www.mdpi.com/1999-4915/10/11/595Viruses, Vol. 10, Pages 594: Impact of Two Reoviruses and Their Coinfection on the Rice RNAi System and vsiRNA Productionhttp://www.mdpi.com/1999-4915/10/11/594
Both Southern rice black-streaked dwarf virus (SRBSDV) and Rice ragged stunt virus (RRSV) belong to the family Reoviridae, and synergistic infection of these two viruses commonly occurs in the field. This study revealed that both SRBSDV and RRSV affect the RNA interference (RNAi) pathway and form different virus-derived interfering RNA (vsiRNA) profiles in rice. Co-infection of rice by SRBSDV and RRSV up-regulated the expression of rice DICER-like (DCL) proteins but down-regulated the expression of rice RNA-dependent RNA polymerases (RDRs), and the accumulation of vsiRNAs of either RBSDV or RRSV was decreased compared with that in singly infected plants. The majority of SRBSDV vsiRNAs were 21 nt or 22 nt in length, whether plants were singly infected with SRBSDV or co-infected with RRSV. On the other hand, the majority of RRSV vsiRNAs were 20 nt, 21 nt, or 22 nt in length, among which those 20 nt in length accounted for the largest proportion; co-infection with SRBSDV further increased the proportion of 20 nt vsiRNAs and decreased the proportion of 21 nt vsiRNAs. Co-infection had no effects on the strand favoritism and hot spots of the vsiRNAs, but changed the bias of the 5&amp;prime; terminal nucleotide significantly. This study provides a reference for further study on the pathogenesis and synergistic mechanism of SRBSDV and RRSV.Viruses, Vol. 10, Pages 594: Impact of Two Reoviruses and Their Coinfection on the Rice RNAi System and vsiRNA Production

Both Southern rice black-streaked dwarf virus (SRBSDV) and Rice ragged stunt virus (RRSV) belong to the family Reoviridae, and synergistic infection of these two viruses commonly occurs in the field. This study revealed that both SRBSDV and RRSV affect the RNA interference (RNAi) pathway and form different virus-derived interfering RNA (vsiRNA) profiles in rice. Co-infection of rice by SRBSDV and RRSV up-regulated the expression of rice DICER-like (DCL) proteins but down-regulated the expression of rice RNA-dependent RNA polymerases (RDRs), and the accumulation of vsiRNAs of either RBSDV or RRSV was decreased compared with that in singly infected plants. The majority of SRBSDV vsiRNAs were 21 nt or 22 nt in length, whether plants were singly infected with SRBSDV or co-infected with RRSV. On the other hand, the majority of RRSV vsiRNAs were 20 nt, 21 nt, or 22 nt in length, among which those 20 nt in length accounted for the largest proportion; co-infection with SRBSDV further increased the proportion of 20 nt vsiRNAs and decreased the proportion of 21 nt vsiRNAs. Co-infection had no effects on the strand favoritism and hot spots of the vsiRNAs, but changed the bias of the 5&amp;prime; terminal nucleotide significantly. This study provides a reference for further study on the pathogenesis and synergistic mechanism of SRBSDV and RRSV.

]]>Impact of Two Reoviruses and Their Coinfection on the Rice RNAi System and vsiRNA ProductionZhanbiao LiTong ZhangXiuqin HuangGuohui Zhoudoi: 10.3390/v10110594Viruses2018-10-30Viruses2018-10-301011Article59410.3390/v10110594http://www.mdpi.com/1999-4915/10/11/594Viruses, Vol. 10, Pages 593: Research Models and Tools for the Identification of Antivirals and Therapeutics against Zika Virus Infectionhttp://www.mdpi.com/1999-4915/10/11/593
Zika virus recently re-emerged and caused global outbreaks mainly in Central Africa, Southeast Asia, the Pacific Islands and in Central and South America. Even though there is a declining trend, the virus continues to spread throughout different geographical regions of the world. Since its re-emergence in 2015, massive advances have been made regarding our understanding of clinical manifestations, epidemiology, genetic diversity, genomic structure and potential therapeutic intervention strategies. Nevertheless, treatment remains a challenge as there is no licensed effective therapy available. This review focuses on the recent advances regarding research models, as well as available experimental tools that can be used for the identification and characterization of potential antiviral targets and therapeutic intervention strategies.Viruses, Vol. 10, Pages 593: Research Models and Tools for the Identification of Antivirals and Therapeutics against Zika Virus Infection

Zika virus recently re-emerged and caused global outbreaks mainly in Central Africa, Southeast Asia, the Pacific Islands and in Central and South America. Even though there is a declining trend, the virus continues to spread throughout different geographical regions of the world. Since its re-emergence in 2015, massive advances have been made regarding our understanding of clinical manifestations, epidemiology, genetic diversity, genomic structure and potential therapeutic intervention strategies. Nevertheless, treatment remains a challenge as there is no licensed effective therapy available. This review focuses on the recent advances regarding research models, as well as available experimental tools that can be used for the identification and characterization of potential antiviral targets and therapeutic intervention strategies.

]]>Research Models and Tools for the Identification of Antivirals and Therapeutics against Zika Virus InfectionMarco P. AlvesNathalie J. VielleVolker ThielStephanie Pfaenderdoi: 10.3390/v10110593Viruses2018-10-30Viruses2018-10-301011Review59310.3390/v10110593http://www.mdpi.com/1999-4915/10/11/593Viruses, Vol. 10, Pages 592: Silvestrol Inhibits Chikungunya Virus Replicationhttp://www.mdpi.com/1999-4915/10/11/592
Silvestrol, a natural compound that is isolated from plants of the genus Aglaia, is a specific inhibitor of the RNA helicase eIF4A, which unwinds RNA secondary structures in 5&amp;prime;-untranslated regions (UTRs) of mRNAs and allows translation. Silvestrol has a broad antiviral activity against multiple RNA virus families. Here, we show that silvestrol inhibits the replication of chikungunya virus (CHIKV), a positive single-stranded RNA virus. Silvestrol delayed the protein synthesis of non-structural (nsPs) and structural proteins, resulting in a delayed innate response to CHIKV infection. Interferon-&amp;alpha; induced STAT1 phosphorylation was not inhibited nor did eIF2&amp;alpha; become phosphorylated 16 h post infection in the presence of silvestrol. In addition, the host protein shut-off induced by CHIKV infection was decreased in silvestrol-treated cells. Silvestrol acts by limiting the amount of nsPs, and thereby reducing CHIKV RNA replication. From our results, we propose that inhibition of the host helicase eIF4A might have potential as a therapeutic strategy to treat CHIKV infections.Viruses, Vol. 10, Pages 592: Silvestrol Inhibits Chikungunya Virus Replication

Silvestrol, a natural compound that is isolated from plants of the genus Aglaia, is a specific inhibitor of the RNA helicase eIF4A, which unwinds RNA secondary structures in 5&amp;prime;-untranslated regions (UTRs) of mRNAs and allows translation. Silvestrol has a broad antiviral activity against multiple RNA virus families. Here, we show that silvestrol inhibits the replication of chikungunya virus (CHIKV), a positive single-stranded RNA virus. Silvestrol delayed the protein synthesis of non-structural (nsPs) and structural proteins, resulting in a delayed innate response to CHIKV infection. Interferon-&amp;alpha; induced STAT1 phosphorylation was not inhibited nor did eIF2&amp;alpha; become phosphorylated 16 h post infection in the presence of silvestrol. In addition, the host protein shut-off induced by CHIKV infection was decreased in silvestrol-treated cells. Silvestrol acts by limiting the amount of nsPs, and thereby reducing CHIKV RNA replication. From our results, we propose that inhibition of the host helicase eIF4A might have potential as a therapeutic strategy to treat CHIKV infections.

]]>Silvestrol Inhibits Chikungunya Virus ReplicationLisa HenssTatjana ScholzArnold GrünwellerBarbara S. Schnierledoi: 10.3390/v10110592Viruses2018-10-30Viruses2018-10-301011Article59210.3390/v10110592http://www.mdpi.com/1999-4915/10/11/592Viruses, Vol. 10, Pages 591: Non-Coding RNAs and Hepatitis C Virus-Induced Hepatocellular Carcinomahttp://www.mdpi.com/1999-4915/10/11/591
Hepatitis C virus (HCV) infection is a worldwide health problem and is one of the main causes of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma (HCC). Despite recent improvements, effective treatments for HCC are still missing and new tools for early detection are needed. Non-coding RNAs (ncRNAs) have emerged as important regulators of gene expression and key players in human carcinogenesis, including HCC. Aberrant expression of ncRNAs is associated with HCC metastasis, invasion, dissemination, and recurrence. This review will focus on the recent advances in ncRNA expression profiles, their dysregulation in HCV-related HCC, and the clinical perspective of ncRNA signatures for the early detection of HCC.Viruses, Vol. 10, Pages 591: Non-Coding RNAs and Hepatitis C Virus-Induced Hepatocellular Carcinoma

Hepatitis C virus (HCV) infection is a worldwide health problem and is one of the main causes of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma (HCC). Despite recent improvements, effective treatments for HCC are still missing and new tools for early detection are needed. Non-coding RNAs (ncRNAs) have emerged as important regulators of gene expression and key players in human carcinogenesis, including HCC. Aberrant expression of ncRNAs is associated with HCC metastasis, invasion, dissemination, and recurrence. This review will focus on the recent advances in ncRNA expression profiles, their dysregulation in HCV-related HCC, and the clinical perspective of ncRNA signatures for the early detection of HCC.

]]>Non-Coding RNAs and Hepatitis C Virus-Induced Hepatocellular CarcinomaMarie-Laure PlissonnierKatharina HerzogMassimo LevreroMirjam B. Zeiseldoi: 10.3390/v10110591Viruses2018-10-30Viruses2018-10-301011Review59110.3390/v10110591http://www.mdpi.com/1999-4915/10/11/591Viruses, Vol. 10, Pages 590: Heterologous Replicase from Cucumoviruses can Replicate Viral RNAs, but is Defective in Transcribing Subgenomic RNA4A or Facilitating Viral Movementhttp://www.mdpi.com/1999-4915/10/11/590
Interspecific exchange of RNA1 or RNA2 between the cucumoviruses cucumber mosaic virus (CMV) and tomato aspermy virus (TAV) was reported to be non-viable in plants previously. Here we investigated viability of the reassortants between CMV and TAV in Nicotiana benthamiana plants by Agrobacterium-mediated viral inoculation. The reassortants were composed of CMV RNA1 and TAV RNA2 plus RNA3 replicated in the inoculated leaves, while they were defective in viral systemic movement at the early stage of infection. Interestingly, the reassortant containing TAV RNA1 and CMV RNA2 and RNA3 infected plants systemically, but produced RNA4A (the RNA2 subgenome) at an undetectable level. The defect in production of RNA4A was due to the 1a protein encoded by TAV RNA1, and partially restored by replacing the C-terminus (helicase domain) in TAV 1a with that of CMV 1a. Collectively, exchange of the replicase components between CMV and TAV was acceptable for viral replication, but was defective in either directing transcription of subgenomic RNA4A or facilitating viral long-distance movement. Our finding may shed some light on evolution of subgenomic RNA4A in the family Bromoviridae.Viruses, Vol. 10, Pages 590: Heterologous Replicase from Cucumoviruses can Replicate Viral RNAs, but is Defective in Transcribing Subgenomic RNA4A or Facilitating Viral Movement

Interspecific exchange of RNA1 or RNA2 between the cucumoviruses cucumber mosaic virus (CMV) and tomato aspermy virus (TAV) was reported to be non-viable in plants previously. Here we investigated viability of the reassortants between CMV and TAV in Nicotiana benthamiana plants by Agrobacterium-mediated viral inoculation. The reassortants were composed of CMV RNA1 and TAV RNA2 plus RNA3 replicated in the inoculated leaves, while they were defective in viral systemic movement at the early stage of infection. Interestingly, the reassortant containing TAV RNA1 and CMV RNA2 and RNA3 infected plants systemically, but produced RNA4A (the RNA2 subgenome) at an undetectable level. The defect in production of RNA4A was due to the 1a protein encoded by TAV RNA1, and partially restored by replacing the C-terminus (helicase domain) in TAV 1a with that of CMV 1a. Collectively, exchange of the replicase components between CMV and TAV was acceptable for viral replication, but was defective in either directing transcription of subgenomic RNA4A or facilitating viral long-distance movement. Our finding may shed some light on evolution of subgenomic RNA4A in the family Bromoviridae.

]]>Heterologous Replicase from Cucumoviruses can Replicate Viral RNAs, but is Defective in Transcribing Subgenomic RNA4A or Facilitating Viral MovementShuangyu GaoJinda LuXiaodong ChengZhouhang GuQiansheng LiaoZhiyou Dudoi: 10.3390/v10110590Viruses2018-10-28Viruses2018-10-281011Article59010.3390/v10110590http://www.mdpi.com/1999-4915/10/11/590Viruses, Vol. 10, Pages 589: Sclerotinia minor Endornavirus 1, a Novel Pathogenicity Debilitation-Associated Mycovirus with a Wide Spectrum of Horizontal Transmissibilityhttp://www.mdpi.com/1999-4915/10/11/589
Sclerotinia minor is a phytopathogenic fungus causing sclerotinia blight on many economically important crops. Here, we have characterized the biological and molecular properties of a novel endornavirus, Sclerotinia minor endornavirus 1 (SmEV1), isolated from the hypovirulent strain LC22 of S. minor. The genome of SmEV1 is 12,626 bp long with a single, large open reading frame (ORF), coding for a putative protein of 4020 amino acids. The putative protein contains cysteine-rich region (CRR), viral methyltransferase (MTR), putative DEXDc, viral helicase (Hel), and RNA-dependent RNA polymerase (RdRp) domains. The putative protein and the conserved domains are phylogenetically related to endornaviruses. SmEV1 does not contain a site-specific nick characteristic of most previously described endornaviruses. Hypovirulence and associated traits of strain LC22 and SmEV1 were readily cotransmitted horizontally via hyphal contact to isolates of different vegetative compatibility groups of S. minor. Additionally, SmEV1 in strain LC22 was found capable of being transmitted vertically through sclerotia. Furthermore, mycelium fragments of hypovirulent strain LC22 have a protective activity against attack by S. minor. Taken together, we concluded that SmEV1 is a novel hypovirulence-associated mycovirus with a wide spectrum of transmissibility, and has potential for biological control (virocontrol) of diseases caused by S. minor.Viruses, Vol. 10, Pages 589: Sclerotinia minor Endornavirus 1, a Novel Pathogenicity Debilitation-Associated Mycovirus with a Wide Spectrum of Horizontal Transmissibility

Sclerotinia minor is a phytopathogenic fungus causing sclerotinia blight on many economically important crops. Here, we have characterized the biological and molecular properties of a novel endornavirus, Sclerotinia minor endornavirus 1 (SmEV1), isolated from the hypovirulent strain LC22 of S. minor. The genome of SmEV1 is 12,626 bp long with a single, large open reading frame (ORF), coding for a putative protein of 4020 amino acids. The putative protein contains cysteine-rich region (CRR), viral methyltransferase (MTR), putative DEXDc, viral helicase (Hel), and RNA-dependent RNA polymerase (RdRp) domains. The putative protein and the conserved domains are phylogenetically related to endornaviruses. SmEV1 does not contain a site-specific nick characteristic of most previously described endornaviruses. Hypovirulence and associated traits of strain LC22 and SmEV1 were readily cotransmitted horizontally via hyphal contact to isolates of different vegetative compatibility groups of S. minor. Additionally, SmEV1 in strain LC22 was found capable of being transmitted vertically through sclerotia. Furthermore, mycelium fragments of hypovirulent strain LC22 have a protective activity against attack by S. minor. Taken together, we concluded that SmEV1 is a novel hypovirulence-associated mycovirus with a wide spectrum of transmissibility, and has potential for biological control (virocontrol) of diseases caused by S. minor.

]]>Sclerotinia minor Endornavirus 1, a Novel Pathogenicity Debilitation-Associated Mycovirus with a Wide Spectrum of Horizontal TransmissibilityDan YangMingde WuJing ZhangWeidong ChenGuoqing LiLong Yangdoi: 10.3390/v10110589Viruses2018-10-27Viruses2018-10-271011Article58910.3390/v10110589http://www.mdpi.com/1999-4915/10/11/589Viruses, Vol. 10, Pages 588: High Incidence of Lysogeny in the Oxygen Minimum Zones of the Arabian Sea (Southwest Coast of India)http://www.mdpi.com/1999-4915/10/11/588
Though microbial processes in the oxygen minimum zones (OMZs) of the Arabian Sea (AS) are well documented, prokaryote-virus interactions are less known. The present study was carried out to determine the potential physico-chemical factors influencing viral abundances and their life strategies (lytic and lysogenic) along the vertical gradient in the OMZ of the AS (southwest coast of India). Water samples were collected during the southwest monsoon (SWM) season in two consecutive years (2015 and 2016) from different depths, namely, the surface layer, secondary chlorophyll a maxima (~30&amp;ndash;40 m), oxycline (~70&amp;ndash;80 m), and hypoxic/suboxic layers (~200&amp;ndash;350 m). The high viral abundances observed in oxygenated surface waters (mean &amp;plusmn; SD = 6.1 &amp;plusmn; 3.4 &amp;times; 106 viral-like particles (VLPs) mL&amp;minus;1), drastically decreased with depth in the oxycline region (1.2 &amp;plusmn; 0.5 &amp;times; 106 VLPs mL&amp;minus;1) and hypoxic/suboxic waters (0.3 &amp;plusmn; 0.3 &amp;times; 106 VLPs mL&amp;minus;1). Virus to prokaryote ratio fluctuated in the mixed layer (~10) and declined significantly (p &amp;lt; 0.001) to 1 in the hypoxic layer. Viral production (VP) and frequency of virus infected cells (FIC) were maximum in the surface and minimum in the oxycline layer, whereas the viral lysis was undetectable in the suboxic/hypoxic layer. The detection of a high percentage of lysogeny in suboxic (48%) and oxycline zones (9&amp;ndash;24%), accompanied by undetectable rates of lytic viral infection support the hypothesis that lysogeny may represent the major survival strategy for viruses in unproductive or harsh nutrient/host conditions in deoxygenated waters.Viruses, Vol. 10, Pages 588: High Incidence of Lysogeny in the Oxygen Minimum Zones of the Arabian Sea (Southwest Coast of India)

Though microbial processes in the oxygen minimum zones (OMZs) of the Arabian Sea (AS) are well documented, prokaryote-virus interactions are less known. The present study was carried out to determine the potential physico-chemical factors influencing viral abundances and their life strategies (lytic and lysogenic) along the vertical gradient in the OMZ of the AS (southwest coast of India). Water samples were collected during the southwest monsoon (SWM) season in two consecutive years (2015 and 2016) from different depths, namely, the surface layer, secondary chlorophyll a maxima (~30&amp;ndash;40 m), oxycline (~70&amp;ndash;80 m), and hypoxic/suboxic layers (~200&amp;ndash;350 m). The high viral abundances observed in oxygenated surface waters (mean &amp;plusmn; SD = 6.1 &amp;plusmn; 3.4 &amp;times; 106 viral-like particles (VLPs) mL&amp;minus;1), drastically decreased with depth in the oxycline region (1.2 &amp;plusmn; 0.5 &amp;times; 106 VLPs mL&amp;minus;1) and hypoxic/suboxic waters (0.3 &amp;plusmn; 0.3 &amp;times; 106 VLPs mL&amp;minus;1). Virus to prokaryote ratio fluctuated in the mixed layer (~10) and declined significantly (p &amp;lt; 0.001) to 1 in the hypoxic layer. Viral production (VP) and frequency of virus infected cells (FIC) were maximum in the surface and minimum in the oxycline layer, whereas the viral lysis was undetectable in the suboxic/hypoxic layer. The detection of a high percentage of lysogeny in suboxic (48%) and oxycline zones (9&amp;ndash;24%), accompanied by undetectable rates of lytic viral infection support the hypothesis that lysogeny may represent the major survival strategy for viruses in unproductive or harsh nutrient/host conditions in deoxygenated waters.

]]>High Incidence of Lysogeny in the Oxygen Minimum Zones of the Arabian Sea (Southwest Coast of India)Ammini ParvathiVijayan JasnaSreekumar AparnaAngia Sriram Pradeep RamVijaya Krishna AswathyKizhakkeppat K. BalachandranKallungal Ravunnikutty MuraleedharanDayana MathewTelesphore Sime-Ngandodoi: 10.3390/v10110588Viruses2018-10-27Viruses2018-10-271011Article58810.3390/v10110588http://www.mdpi.com/1999-4915/10/11/588Viruses, Vol. 10, Pages 587: Innate Immunity Activation and RNAi Interplay in Citrus Exocortis Viroid—Tomato Pathosystemhttp://www.mdpi.com/1999-4915/10/11/587
Although viroids are the smallest and simplest plant pathogens known, the molecular mechanisms underlying their pathogenesis remain unclear. To unravel these mechanisms, a dual approach was implemented consisting of in silico identification of potential tomato silencing targets of pospiviroids, and the experimental validation of these targets through the sequencing of small RNAs and RNA ends extracted from tomatoes infected with a severe isolate of Citrus exocortis viroid (CEVd). The generated RNA ends were also used to monitor the differentially-expressed genes. These analyses showed that when CEVd symptoms are well established: (i) CEVd are degraded by at least three Dicer-like (DCL) proteins and possibly by RNA-induced silencing complex (RISC), (ii) five different mRNAs are partially degraded through post-transcriptional gene silencing (PTGS), including argonaute 2a, which is further degraded in phasiRNAs, (iii) Dicer-like 2b and 2d are both upregulated and degraded in phasiRNAs, and (iv) CEVd infection induced a significant shift in gene expression allowing to explain the usual symptoms of pospiviroids on tomato and to demonstrate the constant activation of host innate immunity and systemic acquired resistance (SAR) by these pathogenic RNAs. Finally, based on in silico analysis, potential immunity receptor candidates of viroid-derived RNAs are suggested.Viruses, Vol. 10, Pages 587: Innate Immunity Activation and RNAi Interplay in Citrus Exocortis Viroid—Tomato Pathosystem

Although viroids are the smallest and simplest plant pathogens known, the molecular mechanisms underlying their pathogenesis remain unclear. To unravel these mechanisms, a dual approach was implemented consisting of in silico identification of potential tomato silencing targets of pospiviroids, and the experimental validation of these targets through the sequencing of small RNAs and RNA ends extracted from tomatoes infected with a severe isolate of Citrus exocortis viroid (CEVd). The generated RNA ends were also used to monitor the differentially-expressed genes. These analyses showed that when CEVd symptoms are well established: (i) CEVd are degraded by at least three Dicer-like (DCL) proteins and possibly by RNA-induced silencing complex (RISC), (ii) five different mRNAs are partially degraded through post-transcriptional gene silencing (PTGS), including argonaute 2a, which is further degraded in phasiRNAs, (iii) Dicer-like 2b and 2d are both upregulated and degraded in phasiRNAs, and (iv) CEVd infection induced a significant shift in gene expression allowing to explain the usual symptoms of pospiviroids on tomato and to demonstrate the constant activation of host innate immunity and systemic acquired resistance (SAR) by these pathogenic RNAs. Finally, based on in silico analysis, potential immunity receptor candidates of viroid-derived RNAs are suggested.

]]>Innate Immunity Activation and RNAi Interplay in Citrus Exocortis Viroid—Tomato PathosystemThibaut OlivierClaude Bragarddoi: 10.3390/v10110587Viruses2018-10-26Viruses2018-10-261011Article58710.3390/v10110587http://www.mdpi.com/1999-4915/10/11/587Viruses, Vol. 10, Pages 585: The Interaction Dynamics of Two Potato Leafroll Virus Movement Proteins Affects Their Localization to the Outer Membranes of Mitochondria and Plastidshttp://www.mdpi.com/1999-4915/10/11/585
The Luteoviridae is an agriculturally important family of viruses whose replication and transport are restricted to plant phloem. Their genomes encode for four proteins that regulate viral movement. These include two structural proteins that make up the capsid and two non-structural proteins known as P3a and P17. Little is known about how these proteins interact with each other and the host to coordinate virus movement within and between cells. We used quantitative, affinity purification-mass spectrometry to show that the P3a protein of Potato leafroll virus complexes with virus and that this interaction is partially dependent on P17. Bimolecular complementation assays (BiFC) were used to validate that P3a and P17 self-interact as well as directly interact with each other. Co-localization with fluorescent-based organelle markers demonstrates that P3a directs P17 to the mitochondrial outer membrane while P17 regulates the localization of the P3a-P17 heterodimer to plastids. Residues in the C-terminus of P3a were shown to regulate P3a association with host mitochondria by using mutational analysis and also varying BiFC tag orientation. Collectively, our work reveals that the PLRV movement proteins play a game of intracellular hopscotch along host organelles to transport the virus to the cell periphery.Viruses, Vol. 10, Pages 585: The Interaction Dynamics of Two Potato Leafroll Virus Movement Proteins Affects Their Localization to the Outer Membranes of Mitochondria and Plastids

The Luteoviridae is an agriculturally important family of viruses whose replication and transport are restricted to plant phloem. Their genomes encode for four proteins that regulate viral movement. These include two structural proteins that make up the capsid and two non-structural proteins known as P3a and P17. Little is known about how these proteins interact with each other and the host to coordinate virus movement within and between cells. We used quantitative, affinity purification-mass spectrometry to show that the P3a protein of Potato leafroll virus complexes with virus and that this interaction is partially dependent on P17. Bimolecular complementation assays (BiFC) were used to validate that P3a and P17 self-interact as well as directly interact with each other. Co-localization with fluorescent-based organelle markers demonstrates that P3a directs P17 to the mitochondrial outer membrane while P17 regulates the localization of the P3a-P17 heterodimer to plastids. Residues in the C-terminus of P3a were shown to regulate P3a association with host mitochondria by using mutational analysis and also varying BiFC tag orientation. Collectively, our work reveals that the PLRV movement proteins play a game of intracellular hopscotch along host organelles to transport the virus to the cell periphery.

]]>The Interaction Dynamics of Two Potato Leafroll Virus Movement Proteins Affects Their Localization to the Outer Membranes of Mitochondria and PlastidsStacy L. DeBlasioYi XuRichard S. JohnsonAna Rita RebeloMichael J. MacCossStewart M. GrayMichelle Heckdoi: 10.3390/v10110585Viruses2018-10-26Viruses2018-10-261011Article58510.3390/v10110585http://www.mdpi.com/1999-4915/10/11/585Viruses, Vol. 10, Pages 586: New Insights into the Evolutionary and Genomic Landscape of Molluscum Contagiosum Virus (MCV) based on Nine MCV1 and Six MCV2 Complete Genome Sequenceshttp://www.mdpi.com/1999-4915/10/11/586
Molluscum contagiosum virus (MCV) is the sole member of the Molluscipoxvirus genus and the causative agent of molluscum contagiosum (MC), a common skin disease. Although it is an important and frequent human pathogen, its genetic landscape and evolutionary history remain largely unknown. In this study, ten novel complete MCV genome sequences of the two most common MCV genotypes were determined (five MCV1 and five MCV2 sequences) and analyzed together with all MCV complete genomes previously deposited in freely accessible sequence repositories (four MCV1 and a single MCV2). In comparison to MCV1, a higher degree of nucleotide sequence conservation was observed among MCV2 genomes. Large-scale recombination events were identified in two newly assembled MCV1 genomes and one MCV2 genome. One recombination event was located in a newly identified recombinant region of the viral genome, and all previously described recombinant regions were re-identified in at least one novel MCV genome. MCV genes comprising the identified recombinant segments have been previously associated with viral interference with host T-cell and NK-cell immune responses. In conclusion, the two most common MCV genotypes emerged along divergent evolutionary pathways from a common ancestor, and the differences in the heterogeneity of MCV1 and MCV2 populations may be attributed to the strictness of the constraints imposed by the host immune response.Viruses, Vol. 10, Pages 586: New Insights into the Evolutionary and Genomic Landscape of Molluscum Contagiosum Virus (MCV) based on Nine MCV1 and Six MCV2 Complete Genome Sequences

Molluscum contagiosum virus (MCV) is the sole member of the Molluscipoxvirus genus and the causative agent of molluscum contagiosum (MC), a common skin disease. Although it is an important and frequent human pathogen, its genetic landscape and evolutionary history remain largely unknown. In this study, ten novel complete MCV genome sequences of the two most common MCV genotypes were determined (five MCV1 and five MCV2 sequences) and analyzed together with all MCV complete genomes previously deposited in freely accessible sequence repositories (four MCV1 and a single MCV2). In comparison to MCV1, a higher degree of nucleotide sequence conservation was observed among MCV2 genomes. Large-scale recombination events were identified in two newly assembled MCV1 genomes and one MCV2 genome. One recombination event was located in a newly identified recombinant region of the viral genome, and all previously described recombinant regions were re-identified in at least one novel MCV genome. MCV genes comprising the identified recombinant segments have been previously associated with viral interference with host T-cell and NK-cell immune responses. In conclusion, the two most common MCV genotypes emerged along divergent evolutionary pathways from a common ancestor, and the differences in the heterogeneity of MCV1 and MCV2 populations may be attributed to the strictness of the constraints imposed by the host immune response.

]]>New Insights into the Evolutionary and Genomic Landscape of Molluscum Contagiosum Virus (MCV) based on Nine MCV1 and Six MCV2 Complete Genome SequencesTomaž M. ZorecDenis KutnjakLea HošnjakBlanka KušarKatarina TrčkoBoštjan J. KocjanYu LiMiljenko KrižmarićJovan MiljkovićMaja RavnikarMario Poljakdoi: 10.3390/v10110586Viruses2018-10-26Viruses2018-10-261011Article58610.3390/v10110586http://www.mdpi.com/1999-4915/10/11/586Viruses, Vol. 10, Pages 584: Novel Mitoviruses and a Unique Tymo-Like Virus in Hypovirulent and Virulent Strains of the Fusarium Head Blight Fungus, Fusarium boothiihttp://www.mdpi.com/1999-4915/10/11/584
Hypovirulence of phytopathogenic fungi are often conferred by mycovirus(es) infections and for this reason many mycoviruses have been characterized, contributing to a better understanding of virus diversity. In this study, three strains of Fusarium head blight fungus (Fusarium boothii) were isolated from Ethiopian wheats as dsRNA-carrying strains: hypovirulent Ep-BL13 (&amp;gt;10, 3 and 2.5 kbp dsRNAs), and virulent Ep-BL14 and Ep-N28 (3 kbp dsRNA each) strains. The 3 kbp-dsRNAs shared 98% nucleotide identity and have single ORFs encoding a replicase when applied to mitochondrial codon usage. Phylogenetic analysis revealed these were strains of a new species termed Fusarium boothii mitovirus 1 in the genus Mitovirus. The largest and smallest dsRNAs in Ep-BL13 appeared to possess single ORFs and the smaller was originated from the larger by removal of its most middle part. The large dsRNA encoded a replicase sharing the highest amino acid identity (35%) with that of Botrytis virus F, the sole member of the family Gammaflexiviridae. Given that the phylogenetic placement, large genome size, simple genomic and unusual 3&amp;prime;-terminal RNA structures were far different from members in the order Tymovirales, the virus termed Fusarium boothii large flexivirus 1 may form a novel genus and family under the order.Viruses, Vol. 10, Pages 584: Novel Mitoviruses and a Unique Tymo-Like Virus in Hypovirulent and Virulent Strains of the Fusarium Head Blight Fungus, Fusarium boothii

Hypovirulence of phytopathogenic fungi are often conferred by mycovirus(es) infections and for this reason many mycoviruses have been characterized, contributing to a better understanding of virus diversity. In this study, three strains of Fusarium head blight fungus (Fusarium boothii) were isolated from Ethiopian wheats as dsRNA-carrying strains: hypovirulent Ep-BL13 (&amp;gt;10, 3 and 2.5 kbp dsRNAs), and virulent Ep-BL14 and Ep-N28 (3 kbp dsRNA each) strains. The 3 kbp-dsRNAs shared 98% nucleotide identity and have single ORFs encoding a replicase when applied to mitochondrial codon usage. Phylogenetic analysis revealed these were strains of a new species termed Fusarium boothii mitovirus 1 in the genus Mitovirus. The largest and smallest dsRNAs in Ep-BL13 appeared to possess single ORFs and the smaller was originated from the larger by removal of its most middle part. The large dsRNA encoded a replicase sharing the highest amino acid identity (35%) with that of Botrytis virus F, the sole member of the family Gammaflexiviridae. Given that the phylogenetic placement, large genome size, simple genomic and unusual 3&amp;prime;-terminal RNA structures were far different from members in the order Tymovirales, the virus termed Fusarium boothii large flexivirus 1 may form a novel genus and family under the order.

]]>Novel Mitoviruses and a Unique Tymo-Like Virus in Hypovirulent and Virulent Strains of the Fusarium Head Blight Fungus, Fusarium boothiiYukiyoshi MizutaniAdane AbrahamKazuma UesakaHideki KondoHaruhisa SugaNobuhiro SuzukiSotaro Chibadoi: 10.3390/v10110584Viruses2018-10-26Viruses2018-10-261011Article58410.3390/v10110584http://www.mdpi.com/1999-4915/10/11/584Viruses, Vol. 10, Pages 583: Pantoea Bacteriophage vB_PagS_Vid5: A Low-Temperature Siphovirus That Harbors a Cluster of Genes Involved in the Biosynthesis of Archaeosinehttp://www.mdpi.com/1999-4915/10/11/583
A novel low-temperature siphovirus, vB_PagS_Vid5 (Vid5), was isolated in Lithuania using Pantoea agglomerans isolate for the phage propagation. The 61,437 bp genome of Vid5 has a G&amp;ndash;C content of 48.8% and contains 99 probable protein encoding genes and one gene for tRNASer. A comparative sequence analysis revealed that 46 out of 99 Vid5 open reading frames (ORFs) code for unique proteins that have no reliable identity to database entries. In total, 33 Vid5 ORFs were given a putative functional annotation, including those coding for the proteins responsible for virion morphogenesis, phage-host interactions, and DNA metabolism. In addition, a cluster of genes possibly involved in the biosynthesis of 7-deazaguanine derivatives was identified. Notably, one of these genes encodes a putative preQ0/preQ1 transporter, which has never been detected in bacteriophages to date. A proteomic analysis led to the experimental identification of 11 virion proteins, including nine that were predicted by bioinformatics approaches. Based on the phylogenetic analysis, Vid5 cannot be assigned to any genus currently recognized by ICTV, and may represent a new one within the family of Siphoviridae.Viruses, Vol. 10, Pages 583: Pantoea Bacteriophage vB_PagS_Vid5: A Low-Temperature Siphovirus That Harbors a Cluster of Genes Involved in the Biosynthesis of Archaeosine

A novel low-temperature siphovirus, vB_PagS_Vid5 (Vid5), was isolated in Lithuania using Pantoea agglomerans isolate for the phage propagation. The 61,437 bp genome of Vid5 has a G&amp;ndash;C content of 48.8% and contains 99 probable protein encoding genes and one gene for tRNASer. A comparative sequence analysis revealed that 46 out of 99 Vid5 open reading frames (ORFs) code for unique proteins that have no reliable identity to database entries. In total, 33 Vid5 ORFs were given a putative functional annotation, including those coding for the proteins responsible for virion morphogenesis, phage-host interactions, and DNA metabolism. In addition, a cluster of genes possibly involved in the biosynthesis of 7-deazaguanine derivatives was identified. Notably, one of these genes encodes a putative preQ0/preQ1 transporter, which has never been detected in bacteriophages to date. A proteomic analysis led to the experimental identification of 11 virion proteins, including nine that were predicted by bioinformatics approaches. Based on the phylogenetic analysis, Vid5 cannot be assigned to any genus currently recognized by ICTV, and may represent a new one within the family of Siphoviridae.

]]>Pantoea Bacteriophage vB_PagS_Vid5: A Low-Temperature Siphovirus That Harbors a Cluster of Genes Involved in the Biosynthesis of ArchaeosineEugenijus ŠimoliūnasMonika ŠimoliūnienėLaura KalinieneAurelija ZajančkauskaitėMartynas SkapasRolandas MeškysAlgirdas KaupinisMindaugas ValiusLidija Truncaitėdoi: 10.3390/v10110583Viruses2018-10-25Viruses2018-10-251011Article58310.3390/v10110583http://www.mdpi.com/1999-4915/10/11/583Viruses, Vol. 10, Pages 582: Evidence of Human Parvovirus B19 Infection in the Post-Mortem Brain Tissue of the Elderlyhttp://www.mdpi.com/1999-4915/10/11/582
After primary exposure, the human parvovirus B19 (B19V) genome may remain in the central nervous system (CNS), establishing a lifelong latency. The structural characteristics and functions of the infected cells are essential for the virus to complete its life cycle. Although B19V has been detected in the brain tissue by sequencing PCR products, little is known about its in vivo cell tropism and pathogenic potential in the CNS. To detect B19V and investigate the distribution of its target cells in the CNS, we studied brain autopsies of elderly subjects using molecular virology, and optical and electron microscopy methods. Our study detected B19V in brain tissue samples from both encephalopathy and control groups, suggesting virus persistence within the CNS throughout the host&amp;rsquo;s lifetime. It appears that within the CNS, the main target of B19V is oligodendrocytes. The greatest number of B19V-positive oligodendrocytes was found in the white matter of the frontal lobe. The number was significantly lower in the gray matter of the frontal lobe (p = 0.008) and the gray and white matter of the temporal lobes (p &amp;lt; 0.0001). The morphological changes observed in the encephalopathy group, propose a possible B19V involvement in the demyelination process.Viruses, Vol. 10, Pages 582: Evidence of Human Parvovirus B19 Infection in the Post-Mortem Brain Tissue of the Elderly

After primary exposure, the human parvovirus B19 (B19V) genome may remain in the central nervous system (CNS), establishing a lifelong latency. The structural characteristics and functions of the infected cells are essential for the virus to complete its life cycle. Although B19V has been detected in the brain tissue by sequencing PCR products, little is known about its in vivo cell tropism and pathogenic potential in the CNS. To detect B19V and investigate the distribution of its target cells in the CNS, we studied brain autopsies of elderly subjects using molecular virology, and optical and electron microscopy methods. Our study detected B19V in brain tissue samples from both encephalopathy and control groups, suggesting virus persistence within the CNS throughout the host&amp;rsquo;s lifetime. It appears that within the CNS, the main target of B19V is oligodendrocytes. The greatest number of B19V-positive oligodendrocytes was found in the white matter of the frontal lobe. The number was significantly lower in the gray matter of the frontal lobe (p = 0.008) and the gray and white matter of the temporal lobes (p &amp;lt; 0.0001). The morphological changes observed in the encephalopathy group, propose a possible B19V involvement in the demyelination process.

]]>Evidence of Human Parvovirus B19 Infection in the Post-Mortem Brain Tissue of the ElderlySandra SkujaAnda VilmaneSimons SvirskisValerija GromaModra Murovskadoi: 10.3390/v10110582Viruses2018-10-25Viruses2018-10-251011Article58210.3390/v10110582http://www.mdpi.com/1999-4915/10/11/582Viruses, Vol. 10, Pages 581: Elements Involved in the Rsv3-Mediated Extreme Resistance against an Avirulent Strain of Soybean Mosaic Virushttp://www.mdpi.com/1999-4915/10/11/581
Extreme resistance (ER) is a type of R-gene-mediated resistance that rapidly induces a symptomless resistance phenotype, which is different from the phenotypical R-resistance manifested by the programmed cell death, accumulation of reactive oxygen species, and hypersensitive response. The Rsv3 gene in soybean cultivar L29 is responsible for ER against the avirulent strain G5H of soybean mosaic virus (SMV), but is ineffective against the virulent strain G7H. Rsv3-mediated ER is achieved through the rapid accumulation of callose, which arrests SMV-G5H at the point of infection. Callose accumulation, however, may not be the lone mechanism of this ER. Analyses of RNA-seq data obtained from infected soybean plants revealed a rapid induction of the abscisic acid pathway at 8 h post infection (hpi) in response to G5H but not to G7H, which resulted in the down-regulation of transcripts encoding &amp;beta;-1,3 glucanases that degrade callose in G5H-infected but not G7H-infected plants. In addition, parts of the autophagy and the small interfering (si) RNA pathways were temporally up-regulated at 24 hpi in response to G5H but not in response to G7H. The jasmonic acid (JA) pathway and many WRKY factors were clearly up-regulated only in G7H-infected plants. These results suggest that ER against SMV-G5H is achieved through the quick and temporary induction of ABA, autophagy, and the siRNA pathways, which rapidly eliminate G5H. The results also suggest that suppression of the JA pathway in the case of G5H is important for the Rsv3-mediated ER.Viruses, Vol. 10, Pages 581: Elements Involved in the Rsv3-Mediated Extreme Resistance against an Avirulent Strain of Soybean Mosaic Virus

Extreme resistance (ER) is a type of R-gene-mediated resistance that rapidly induces a symptomless resistance phenotype, which is different from the phenotypical R-resistance manifested by the programmed cell death, accumulation of reactive oxygen species, and hypersensitive response. The Rsv3 gene in soybean cultivar L29 is responsible for ER against the avirulent strain G5H of soybean mosaic virus (SMV), but is ineffective against the virulent strain G7H. Rsv3-mediated ER is achieved through the rapid accumulation of callose, which arrests SMV-G5H at the point of infection. Callose accumulation, however, may not be the lone mechanism of this ER. Analyses of RNA-seq data obtained from infected soybean plants revealed a rapid induction of the abscisic acid pathway at 8 h post infection (hpi) in response to G5H but not to G7H, which resulted in the down-regulation of transcripts encoding &amp;beta;-1,3 glucanases that degrade callose in G5H-infected but not G7H-infected plants. In addition, parts of the autophagy and the small interfering (si) RNA pathways were temporally up-regulated at 24 hpi in response to G5H but not in response to G7H. The jasmonic acid (JA) pathway and many WRKY factors were clearly up-regulated only in G7H-infected plants. These results suggest that ER against SMV-G5H is achieved through the quick and temporary induction of ABA, autophagy, and the siRNA pathways, which rapidly eliminate G5H. The results also suggest that suppression of the JA pathway in the case of G5H is important for the Rsv3-mediated ER.

]]>Elements Involved in the Rsv3-Mediated Extreme Resistance against an Avirulent Strain of Soybean Mosaic VirusMazen AlazemKuan-Chieh TsengWen-Chi ChangJang-Kyun SeoKook-Hyung Kimdoi: 10.3390/v10110581Viruses2018-10-24Viruses2018-10-241011Article58110.3390/v10110581http://www.mdpi.com/1999-4915/10/11/581Viruses, Vol. 10, Pages 580: Herbal Gel Formulation Developed for Anti-Human Immunodeficiency Virus (HIV)-1 Activity Also Inhibits In Vitro HSV-2 Infectionhttp://www.mdpi.com/1999-4915/10/11/580
Herpes simplex virus-2 (HSV-2) infection is the most common cause of genital ulcers. The impact of ulcers also demonstrates a strong link to the human immunodeficiency virus (HIV) infection. Complications, drug resistance, and side-effects of anti-viral drugs make the treatment of HSV-2 infection challenging. Herbal medicines have shown potential against HSV-2 and HIV infections. In this context, polyherbal gel formulation comprising 50% ethanolic extracts from Acacia catechu, Lagerstroemia speciosa, Terminalia chebula and Phyllanthus emblica has been developed. The gel formulation significantly exhibited virucidal activity against both HIV-1 and HSV-2 infections with IC50, 55.93 &amp;plusmn; 5.30 &amp;micro;g/mL and 27.26 &amp;plusmn; 4.87 &amp;micro;g/mL, respectively. It also inhibited HSV-2 attachment and penetration to the Vero cells with an IC50 = 46.55 &amp;plusmn; 1.25 &amp;micro;g/mL and 54.94 &amp;plusmn; 2.52 &amp;micro;g/mL respectively, which were significantly lower than acyclovir. However, acyclovir is more potent in post-infection assay with an IC50 = 0.065 &amp;plusmn; 0.01 &amp;micro;g/mL whereas gel formulation showed an IC50 = 469.05 &amp;plusmn; 16.65 &amp;micro;g/mL under similar conditions. Gel formulation showed no inhibitory effect on the viability of lactobacilli, human vaginal keratinocyte cells (Vk2/E6E7), and the integrity of the Caco-2 cells monolayer. Gel formulation did not lead to any significant increase in the secretion of pro-inflammatory cytokines and mutagenic index. The proposed gel formulation may be a promising candidate microbicide for the prevention of sexually transmitted HIV-1 and HSV-2.Viruses, Vol. 10, Pages 580: Herbal Gel Formulation Developed for Anti-Human Immunodeficiency Virus (HIV)-1 Activity Also Inhibits In Vitro HSV-2 Infection

Herpes simplex virus-2 (HSV-2) infection is the most common cause of genital ulcers. The impact of ulcers also demonstrates a strong link to the human immunodeficiency virus (HIV) infection. Complications, drug resistance, and side-effects of anti-viral drugs make the treatment of HSV-2 infection challenging. Herbal medicines have shown potential against HSV-2 and HIV infections. In this context, polyherbal gel formulation comprising 50% ethanolic extracts from Acacia catechu, Lagerstroemia speciosa, Terminalia chebula and Phyllanthus emblica has been developed. The gel formulation significantly exhibited virucidal activity against both HIV-1 and HSV-2 infections with IC50, 55.93 &amp;plusmn; 5.30 &amp;micro;g/mL and 27.26 &amp;plusmn; 4.87 &amp;micro;g/mL, respectively. It also inhibited HSV-2 attachment and penetration to the Vero cells with an IC50 = 46.55 &amp;plusmn; 1.25 &amp;micro;g/mL and 54.94 &amp;plusmn; 2.52 &amp;micro;g/mL respectively, which were significantly lower than acyclovir. However, acyclovir is more potent in post-infection assay with an IC50 = 0.065 &amp;plusmn; 0.01 &amp;micro;g/mL whereas gel formulation showed an IC50 = 469.05 &amp;plusmn; 16.65 &amp;micro;g/mL under similar conditions. Gel formulation showed no inhibitory effect on the viability of lactobacilli, human vaginal keratinocyte cells (Vk2/E6E7), and the integrity of the Caco-2 cells monolayer. Gel formulation did not lead to any significant increase in the secretion of pro-inflammatory cytokines and mutagenic index. The proposed gel formulation may be a promising candidate microbicide for the prevention of sexually transmitted HIV-1 and HSV-2.

]]>Herbal Gel Formulation Developed for Anti-Human Immunodeficiency Virus (HIV)-1 Activity Also Inhibits In Vitro HSV-2 InfectionNripendra Nath MishraAjay KesharwaniAakanksha AgarwalSuja Kizhiyedath PolachiraReshmi NairSatish Kumar Guptadoi: 10.3390/v10110580Viruses2018-10-24Viruses2018-10-241011Article58010.3390/v10110580http://www.mdpi.com/1999-4915/10/11/580Viruses, Vol. 10, Pages 579: Controlled Disassembly and Purification of Functional Viral Subassemblies Using Asymmetrical Flow Field-Flow Fractionation (AF4)http://www.mdpi.com/1999-4915/10/11/579
Viruses protect their genomes by enclosing them into protein capsids that sometimes contain lipid bilayers that either reside above or below the protein layer. Controlled dissociation of virions provides important information on virion composition, interactions, and stoichiometry of virion components, as well as their possible role in virus life cycles. Dissociation of viruses can be achieved by using various chemicals, enzymatic treatments, and incubation conditions. Asymmetrical flow field-flow fractionation (AF4) is a gentle method where the separation is based on size. Here, we applied AF4 for controlled dissociation of enveloped bacteriophage &amp;phi;6. Our results indicate that AF4 can be used to assay the efficiency of the dissociation process and to purify functional subviral particles.Viruses, Vol. 10, Pages 579: Controlled Disassembly and Purification of Functional Viral Subassemblies Using Asymmetrical Flow Field-Flow Fractionation (AF4)

Viruses protect their genomes by enclosing them into protein capsids that sometimes contain lipid bilayers that either reside above or below the protein layer. Controlled dissociation of virions provides important information on virion composition, interactions, and stoichiometry of virion components, as well as their possible role in virus life cycles. Dissociation of viruses can be achieved by using various chemicals, enzymatic treatments, and incubation conditions. Asymmetrical flow field-flow fractionation (AF4) is a gentle method where the separation is based on size. Here, we applied AF4 for controlled dissociation of enveloped bacteriophage &amp;phi;6. Our results indicate that AF4 can be used to assay the efficiency of the dissociation process and to purify functional subviral particles.

]]>Controlled Disassembly and Purification of Functional Viral Subassemblies Using Asymmetrical Flow Field-Flow Fractionation (AF4)Katri EskelinMinna M. Poranendoi: 10.3390/v10110579Viruses2018-10-23Viruses2018-10-231011Article57910.3390/v10110579http://www.mdpi.com/1999-4915/10/11/579Viruses, Vol. 10, Pages 578: Identification and Molecular Characterization of a Novel Partitivirus from Trichoderma atroviride NFCF394http://www.mdpi.com/1999-4915/10/11/578
An increasing number of novel mycoviruses have been described in fungi. Here, we report the molecular characteristics of a novel bisegmented double-stranded RNA (dsRNA) virus from the fungus Trichoderma atroviride NFCF394. We designated this mycovirus as Trichoderma atroviride partitivirus 1 (TaPV1). Electron micrographs of negatively stained, purified viral particles showed an isometric structure approximately of 30 nm in diameter. The larger segment (dsRNA1) of the TaPV1 genome comprised 2023 bp and contained a single open reading frame (ORF) encoding 614 amino acid (AA) residues of RNA-dependent RNA polymerase (RdRp). The smaller segment (dsRNA2) consisted of 2012 bp with a single ORF encoding 577 AA residues of capsid protein (CP). The phylogenetic analysis, based on deduced amino acid sequences of RdRp and CP, indicated that TaPV1 is a new member of the genus Alphapartitivirus in the family Partitiviridae. Virus-cured isogenic strains did not show significant changes in colony morphology. In addition, no changes in the enzymatic activities of &amp;beta;-1,3-glucanase and chitinase were observed in virus-cured strains. To the best of our knowledge, this is the first report of an Alphapartitivirus in T. atroviride.Viruses, Vol. 10, Pages 578: Identification and Molecular Characterization of a Novel Partitivirus from Trichoderma atroviride NFCF394

An increasing number of novel mycoviruses have been described in fungi. Here, we report the molecular characteristics of a novel bisegmented double-stranded RNA (dsRNA) virus from the fungus Trichoderma atroviride NFCF394. We designated this mycovirus as Trichoderma atroviride partitivirus 1 (TaPV1). Electron micrographs of negatively stained, purified viral particles showed an isometric structure approximately of 30 nm in diameter. The larger segment (dsRNA1) of the TaPV1 genome comprised 2023 bp and contained a single open reading frame (ORF) encoding 614 amino acid (AA) residues of RNA-dependent RNA polymerase (RdRp). The smaller segment (dsRNA2) consisted of 2012 bp with a single ORF encoding 577 AA residues of capsid protein (CP). The phylogenetic analysis, based on deduced amino acid sequences of RdRp and CP, indicated that TaPV1 is a new member of the genus Alphapartitivirus in the family Partitiviridae. Virus-cured isogenic strains did not show significant changes in colony morphology. In addition, no changes in the enzymatic activities of &amp;beta;-1,3-glucanase and chitinase were observed in virus-cured strains. To the best of our knowledge, this is the first report of an Alphapartitivirus in T. atroviride.

]]>Identification and Molecular Characterization of a Novel Partitivirus from Trichoderma atroviride NFCF394Jeesun ChunHan-Eul YangDae-Hyuk Kimdoi: 10.3390/v10110578Viruses2018-10-23Viruses2018-10-231011Article57810.3390/v10110578http://www.mdpi.com/1999-4915/10/11/578Viruses, Vol. 10, Pages 577: Biodiversity of Streptococcus thermophilus Phages in Global Dairy Fermentationshttp://www.mdpi.com/1999-4915/10/10/577
Streptococcus thermophilus strains are among the most widely employed starter cultures in dairy fermentations, second only to those of Lactococcus lactis. The extensive application of this species provides considerable opportunity for the proliferation of its infecting (bacterio)phages. Until recently, dairy streptococcal phages were classified into two groups (cos and pac groups), while more recently, two additional groups have been identified (5093 and 987 groups). This highlights the requirement for consistent monitoring of phage populations in the industry. Here, we report a survey of 35 samples of whey derived from 27 dairy fermentation facilities in ten countries against a panel of S. thermophilus strains. This culminated in the identification of 172 plaque isolates, which were characterized by multiplex PCR, restriction fragment length polymorphism analysis, and host range profiling. Based on this characterisation, 39 distinct isolates representing all four phage groups were selected for genome sequencing. Genetic diversity was observed among the cos isolates and correlations between receptor binding protein phylogeny and host range were also clear within this phage group. The 987 phages isolated within this study shared high levels of sequence similarity, yet displayed reduced levels of similarity to those identified in previous studies, indicating that they are subject to ongoing genetic diversification.Viruses, Vol. 10, Pages 577: Biodiversity of Streptococcus thermophilus Phages in Global Dairy Fermentations

Streptococcus thermophilus strains are among the most widely employed starter cultures in dairy fermentations, second only to those of Lactococcus lactis. The extensive application of this species provides considerable opportunity for the proliferation of its infecting (bacterio)phages. Until recently, dairy streptococcal phages were classified into two groups (cos and pac groups), while more recently, two additional groups have been identified (5093 and 987 groups). This highlights the requirement for consistent monitoring of phage populations in the industry. Here, we report a survey of 35 samples of whey derived from 27 dairy fermentation facilities in ten countries against a panel of S. thermophilus strains. This culminated in the identification of 172 plaque isolates, which were characterized by multiplex PCR, restriction fragment length polymorphism analysis, and host range profiling. Based on this characterisation, 39 distinct isolates representing all four phage groups were selected for genome sequencing. Genetic diversity was observed among the cos isolates and correlations between receptor binding protein phylogeny and host range were also clear within this phage group. The 987 phages isolated within this study shared high levels of sequence similarity, yet displayed reduced levels of similarity to those identified in previous studies, indicating that they are subject to ongoing genetic diversification.

]]>Biodiversity of Streptococcus thermophilus Phages in Global Dairy FermentationsKatherine LavelleInes MartinezHorst NeveGabriele A. LugliCharles M. A. P. FranzMarco VenturaFabio dal BelloDouwe van SinderenJennifer Mahonydoi: 10.3390/v10100577Viruses2018-10-22Viruses2018-10-221010Article57710.3390/v10100577http://www.mdpi.com/1999-4915/10/10/577Viruses, Vol. 10, Pages 576: Gene Gangs of the Chloroviruses: Conserved Clusters of Collinear Monocistronic Geneshttp://www.mdpi.com/1999-4915/10/10/576
Chloroviruses (family Phycodnaviridae) are dsDNA viruses found throughout the world’s inland waters. The open reading frames in the genomes of 41 sequenced chloroviruses (330 ± 40 kbp each) representing three virus types were analyzed for evidence of evolutionarily conserved local genomic “contexts”, the organization of biological information into units of a scale larger than a gene. Despite a general loss of synteny between virus types, we informatically detected a highly conserved genomic context defined by groups of three or more genes that we have termed “gene gangs”. Unlike previously described local genomic contexts, the definition of gene gangs requires only that member genes be consistently co-localized and are not constrained by strand, regulatory sites, or intervening sequences (and therefore represent a new type of conserved structural genomic element). An analysis of functional annotations and transcriptomic data suggests that some of the gene gangs may organize genes involved in specific biochemical processes, but that this organization does not involve their coordinated expression.Viruses, Vol. 10, Pages 576: Gene Gangs of the Chloroviruses: Conserved Clusters of Collinear Monocistronic Genes

Chloroviruses (family Phycodnaviridae) are dsDNA viruses found throughout the world’s inland waters. The open reading frames in the genomes of 41 sequenced chloroviruses (330 ± 40 kbp each) representing three virus types were analyzed for evidence of evolutionarily conserved local genomic “contexts”, the organization of biological information into units of a scale larger than a gene. Despite a general loss of synteny between virus types, we informatically detected a highly conserved genomic context defined by groups of three or more genes that we have termed “gene gangs”. Unlike previously described local genomic contexts, the definition of gene gangs requires only that member genes be consistently co-localized and are not constrained by strand, regulatory sites, or intervening sequences (and therefore represent a new type of conserved structural genomic element). An analysis of functional annotations and transcriptomic data suggests that some of the gene gangs may organize genes involved in specific biochemical processes, but that this organization does not involve their coordinated expression.

]]>Gene Gangs of the Chloroviruses: Conserved Clusters of Collinear Monocistronic GenesPhillip SeitzerAdrien JeanniardFangrui MaJames Van EttenMarc FacciottiDavid Dunigandoi: 10.3390/v10100576Viruses2018-10-20Viruses2018-10-201010Article57610.3390/v10100576http://www.mdpi.com/1999-4915/10/10/576Viruses, Vol. 10, Pages 575: Mason-Pfizer Monkey Virus Envelope Glycoprotein Cycling and Its Vesicular Co-Transport with Immature Particleshttp://www.mdpi.com/1999-4915/10/10/575
The envelope glycoprotein (Env) plays a crucial role in the retroviral life cycle by mediating primary interactions with the host cell. As described previously and expanded on in this paper, Env mediates the trafficking of immature Mason-Pfizer monkey virus (M-PMV) particles to the plasma membrane (PM). Using a panel of labeled RabGTPases as endosomal markers, we identified Env mostly in Rab7a- and Rab9a-positive endosomes. Based on an analysis of the transport of recombinant fluorescently labeled M-PMV Gag and Env proteins, we propose a putative mechanism of the intracellular trafficking of M-PMV Env and immature particles. According to this model, a portion of Env is targeted from the trans-Golgi network (TGN) to Rab7a-positive endosomes. It is then transported to Rab9a-positive endosomes and back to the TGN. It is at the Rab9a vesicles where the immature particles may anchor to the membranes of the Env-containing vesicles, preventing Env recycling to the TGN. These Gag-associated vesicles are then transported to the plasma membrane.Viruses, Vol. 10, Pages 575: Mason-Pfizer Monkey Virus Envelope Glycoprotein Cycling and Its Vesicular Co-Transport with Immature Particles

The envelope glycoprotein (Env) plays a crucial role in the retroviral life cycle by mediating primary interactions with the host cell. As described previously and expanded on in this paper, Env mediates the trafficking of immature Mason-Pfizer monkey virus (M-PMV) particles to the plasma membrane (PM). Using a panel of labeled RabGTPases as endosomal markers, we identified Env mostly in Rab7a- and Rab9a-positive endosomes. Based on an analysis of the transport of recombinant fluorescently labeled M-PMV Gag and Env proteins, we propose a putative mechanism of the intracellular trafficking of M-PMV Env and immature particles. According to this model, a portion of Env is targeted from the trans-Golgi network (TGN) to Rab7a-positive endosomes. It is then transported to Rab9a-positive endosomes and back to the TGN. It is at the Rab9a vesicles where the immature particles may anchor to the membranes of the Env-containing vesicles, preventing Env recycling to the TGN. These Gag-associated vesicles are then transported to the plasma membrane.

]]>Mason-Pfizer Monkey Virus Envelope Glycoprotein Cycling and Its Vesicular Co-Transport with Immature ParticlesPetra Grznárová ProkšováJan LipovJaroslav ZelenkaEric HunterHana LangerováMichaela RumlováTomáš Rumldoi: 10.3390/v10100575Viruses2018-10-20Viruses2018-10-201010Article57510.3390/v10100575http://www.mdpi.com/1999-4915/10/10/575Viruses, Vol. 10, Pages 574: Improved Baculovirus Vectors for Transduction and Gene Expression in Human Pancreatic Islet Cellshttp://www.mdpi.com/1999-4915/10/10/574
Pancreatic islet transplantation is a promising treatment for type 1 diabetes mellitus offering improved glycaemic control by restoring insulin production. Improved human pancreatic islet isolation has led to higher islet transplantation success. However, as many as 50% of islets are lost after transplantation due to immune responses and cellular injury, gene therapy presents a novel strategy to protect pancreatic islets for improved survival post-transplantation. To date, most of the vectors used in clinical trials and gene therapy studies have been derived from mammalian viruses such as adeno-associated or retrovirus. However, baculovirus BacMam vectors provide an attractive and safe alternative. Here, a novel BacMam was constructed containing a frameshift mutation within fp25, which results in virus stocks with higher infectious titres. This improved in vitro transduction when compared to control BacMams. Additionally, incorporating a truncated vesicular stomatitis virus G protein increased transduction efficacy and production of EGFP and BCL2 in human kidney (HK-2) and pancreatic islet &amp;beta; cells (EndoC &amp;beta;H3). Lastly, we have shown that our optimized BacMam vector can deliver and express egfp in intact pancreatic islet cells from human cadaveric donors. These results confirm that BacMam vectors are a viable choice for providing delivery of transgenes to pancreatic islet cells.Viruses, Vol. 10, Pages 574: Improved Baculovirus Vectors for Transduction and Gene Expression in Human Pancreatic Islet Cells

Pancreatic islet transplantation is a promising treatment for type 1 diabetes mellitus offering improved glycaemic control by restoring insulin production. Improved human pancreatic islet isolation has led to higher islet transplantation success. However, as many as 50% of islets are lost after transplantation due to immune responses and cellular injury, gene therapy presents a novel strategy to protect pancreatic islets for improved survival post-transplantation. To date, most of the vectors used in clinical trials and gene therapy studies have been derived from mammalian viruses such as adeno-associated or retrovirus. However, baculovirus BacMam vectors provide an attractive and safe alternative. Here, a novel BacMam was constructed containing a frameshift mutation within fp25, which results in virus stocks with higher infectious titres. This improved in vitro transduction when compared to control BacMams. Additionally, incorporating a truncated vesicular stomatitis virus G protein increased transduction efficacy and production of EGFP and BCL2 in human kidney (HK-2) and pancreatic islet &amp;beta; cells (EndoC &amp;beta;H3). Lastly, we have shown that our optimized BacMam vector can deliver and express egfp in intact pancreatic islet cells from human cadaveric donors. These results confirm that BacMam vectors are a viable choice for providing delivery of transgenes to pancreatic islet cells.

]]>Improved Baculovirus Vectors for Transduction and Gene Expression in Human Pancreatic Islet CellsLeo P. GravesMine AksularRiyadh A. AlakeelyDaniel Ruiz BuckAdam C. ChambersFernanda Murguia-MecaJuan-Jose Plata-MuñozStephen HughesPaul R. V. JohnsonRobert D. PosseeLinda A. Kingdoi: 10.3390/v10100574Viruses2018-10-20Viruses2018-10-201010Article57410.3390/v10100574http://www.mdpi.com/1999-4915/10/10/574Viruses, Vol. 10, Pages 573: βTrCP is Required for HIV-1 Vpu Modulation of CD4, GaLV Env, and BST-2/Tetherinhttp://www.mdpi.com/1999-4915/10/10/573
The Human immunodeficiency virus-1 (HIV-1) accessory protein Vpu modulates numerous proteins, including the host proteins CD4 and BST-2/tetherin. Vpu interacts with the Skp, Cullin, F-Box (SCF) ubiquitin ligase through interactions with the F-Box protein &amp;beta;TrCP (1 and/or 2). This interaction is dependent on phosphorylation of S52,56 in Vpu. Mutation of S52,56, or inhibition of the SCF, abolishes most Vpu activity against CD4 and partly reduces activity against BST-2/tetherin. Recently, Vpu has also been reported to interact with the clathrin adapter proteins AP-1 and AP-2, and these interactions were also found to be required for BST-2/tetherin antagonism in an S52,56 -dependent manner. In assays where HIV-1 is pseudotyped with gibbon ape leukemia virus (GaLV Env), Vpu has also been found to prevent GaLV Env from being incorporated into viral particles, but the mechanism for this antagonism is not fully understood. To clarify the role of the &amp;beta;TrCPs in Vpu function we used CRISPR/Cas9 to generate a clonal cell line lacking both &amp;beta;TrCP-1 and -2. Vpu activity against CD4 and GaLV Env was abolished in this cell line, and activity against BST-2/tetherin reduced significantly. Mutation of the S52,56 residues no longer affected Vpu activity against BST-2/tetherin in this cell line. These data suggest that the primary role of the S52,56 residues in antagonism of CD4, GaLV Env, and BST-2/tetherin is to recruit the SCF/&amp;beta;TrCP ubiquitin ligase.Viruses, Vol. 10, Pages 573: βTrCP is Required for HIV-1 Vpu Modulation of CD4, GaLV Env, and BST-2/Tetherin

The Human immunodeficiency virus-1 (HIV-1) accessory protein Vpu modulates numerous proteins, including the host proteins CD4 and BST-2/tetherin. Vpu interacts with the Skp, Cullin, F-Box (SCF) ubiquitin ligase through interactions with the F-Box protein &amp;beta;TrCP (1 and/or 2). This interaction is dependent on phosphorylation of S52,56 in Vpu. Mutation of S52,56, or inhibition of the SCF, abolishes most Vpu activity against CD4 and partly reduces activity against BST-2/tetherin. Recently, Vpu has also been reported to interact with the clathrin adapter proteins AP-1 and AP-2, and these interactions were also found to be required for BST-2/tetherin antagonism in an S52,56 -dependent manner. In assays where HIV-1 is pseudotyped with gibbon ape leukemia virus (GaLV Env), Vpu has also been found to prevent GaLV Env from being incorporated into viral particles, but the mechanism for this antagonism is not fully understood. To clarify the role of the &amp;beta;TrCPs in Vpu function we used CRISPR/Cas9 to generate a clonal cell line lacking both &amp;beta;TrCP-1 and -2. Vpu activity against CD4 and GaLV Env was abolished in this cell line, and activity against BST-2/tetherin reduced significantly. Mutation of the S52,56 residues no longer affected Vpu activity against BST-2/tetherin in this cell line. These data suggest that the primary role of the S52,56 residues in antagonism of CD4, GaLV Env, and BST-2/tetherin is to recruit the SCF/&amp;beta;TrCP ubiquitin ligase.

]]>βTrCP is Required for HIV-1 Vpu Modulation of CD4, GaLV Env, and BST-2/TetherinYul Eum SongDaniel CyburtTiffany M. LucasDevon A. GregoryTerri D. LyddonMarc C. Johnsondoi: 10.3390/v10100573Viruses2018-10-19Viruses2018-10-191010Article57310.3390/v10100573http://www.mdpi.com/1999-4915/10/10/573Viruses, Vol. 10, Pages 572: The Main Risk Factors of Nipah Disease and Its Risk Analysis in Chinahttp://www.mdpi.com/1999-4915/10/10/572
Nipah disease is a highly fatal zoonosis which is caused by the Nipah virus. The Nipah virus is a BSL-4 virus with fruit bats being its natural host. It is mainly prevalent in Southeast Asia. The virus was first discovered in 1997 in Negeri Sembilan, Malaysia. Currently, it is mainly harmful to pigs and humans with a high mortality rate. This study describes the route of transmission of the Nipah virus in different countries and analyzes the possibility of the primary disease being in China and the method of its transmission to China. The risk factors are analyzed for different susceptible populations to Nipah disease. The aim is to improve people&amp;rsquo;s risk awareness and prevention and control of the disease and reduce its risk of occurring and spreading in China.Viruses, Vol. 10, Pages 572: The Main Risk Factors of Nipah Disease and Its Risk Analysis in China

Nipah disease is a highly fatal zoonosis which is caused by the Nipah virus. The Nipah virus is a BSL-4 virus with fruit bats being its natural host. It is mainly prevalent in Southeast Asia. The virus was first discovered in 1997 in Negeri Sembilan, Malaysia. Currently, it is mainly harmful to pigs and humans with a high mortality rate. This study describes the route of transmission of the Nipah virus in different countries and analyzes the possibility of the primary disease being in China and the method of its transmission to China. The risk factors are analyzed for different susceptible populations to Nipah disease. The aim is to improve people&amp;rsquo;s risk awareness and prevention and control of the disease and reduce its risk of occurring and spreading in China.

]]>The Main Risk Factors of Nipah Disease and Its Risk Analysis in ChinaJiarong YuXinbo LvZijun YangShengbin GaoChangming LiYumei CaiJinming Lidoi: 10.3390/v10100572Viruses2018-10-19Viruses2018-10-191010Review57210.3390/v10100572http://www.mdpi.com/1999-4915/10/10/572Viruses, Vol. 10, Pages 571: Ultrastructural Analysis of Chikungunya Virus Dissemination from the Midgut of the Yellow Fever Mosquito, Aedes aegyptihttp://www.mdpi.com/1999-4915/10/10/571
The transmission cycle of chikungunya virus (CHIKV) requires that mosquito vectors get persistently infected with the virus, following its oral acqsuisition from a vertebrate host. The mosquito midgut is the initial organ that gets infected with orally acquired CHIKV. Following its replication in the midgut epithelium, the virus exits the midgut and infects secondary tissues including the salivary glands before being transmitted to another host. Here, we investigate the pattern of CHIKV dissemination from the midgut of Aedes aegypti at the ultrastructural level. Bloodmeal ingestion caused overstretching of the midgut basal lamina (BL), which was disrupted in areas adjacent to muscles surrounding the midgut as shown by scanning electron microscopy (SEM). Using both transmission electron microscopy (TEM) and focused ion beam scanning electron microscopy (FIB-SEM) to analyze midgut preparations, mature chikungunya (CHIK) virions were found accumulating at the BL and within strands of the BL at 24&amp;ndash;32 h post-infectious bloodmeal (pibm). From 48 h pibm onwards, virions no longer congregated at the BL and became dispersed throughout the basal labyrinth of the epithelial cells. Ingestion of a subsequent, non-infectious bloodmeal caused mature virions to congregate again at the midgut BL. Our study suggests that CHIKV needs a single replication cycle in the midgut epithelium before mature virions directly traverse the midgut BL during a relatively narrow time window, within 48 h pibm.Viruses, Vol. 10, Pages 571: Ultrastructural Analysis of Chikungunya Virus Dissemination from the Midgut of the Yellow Fever Mosquito, Aedes aegypti

The transmission cycle of chikungunya virus (CHIKV) requires that mosquito vectors get persistently infected with the virus, following its oral acqsuisition from a vertebrate host. The mosquito midgut is the initial organ that gets infected with orally acquired CHIKV. Following its replication in the midgut epithelium, the virus exits the midgut and infects secondary tissues including the salivary glands before being transmitted to another host. Here, we investigate the pattern of CHIKV dissemination from the midgut of Aedes aegypti at the ultrastructural level. Bloodmeal ingestion caused overstretching of the midgut basal lamina (BL), which was disrupted in areas adjacent to muscles surrounding the midgut as shown by scanning electron microscopy (SEM). Using both transmission electron microscopy (TEM) and focused ion beam scanning electron microscopy (FIB-SEM) to analyze midgut preparations, mature chikungunya (CHIK) virions were found accumulating at the BL and within strands of the BL at 24&amp;ndash;32 h post-infectious bloodmeal (pibm). From 48 h pibm onwards, virions no longer congregated at the BL and became dispersed throughout the basal labyrinth of the epithelial cells. Ingestion of a subsequent, non-infectious bloodmeal caused mature virions to congregate again at the midgut BL. Our study suggests that CHIKV needs a single replication cycle in the midgut epithelium before mature virions directly traverse the midgut BL during a relatively narrow time window, within 48 h pibm.

]]>Ultrastructural Analysis of Chikungunya Virus Dissemination from the Midgut of the Yellow Fever Mosquito, Aedes aegyptiAsher M. KantorDeAna G. GrantVelmurugan BalaramanTommi A. WhiteAlexander W. E. Franzdoi: 10.3390/v10100571Viruses2018-10-18Viruses2018-10-181010Article57110.3390/v10100571http://www.mdpi.com/1999-4915/10/10/571Viruses, Vol. 10, Pages 568: Rapid Construction of a Replication-Competent Infectious Clone of Human Adenovirus Type 14 by Gibson Assemblyhttp://www.mdpi.com/1999-4915/10/10/568
In 1955, Human adenovirus type 14 (HAdV-B14p) was firstly identified in a military trainee diagnosed as acute respiratory disease (ARD) in the Netherlands. Fifty years later, a genomic variant, HAdV-B14p1, re-emerged in the U.S. and caused large and fatal ARD outbreaks. Subsequently, more and more ARD outbreaks occurred in Canada, the UK, Ireland, and China, in both military and civil settings. To generate a tool for the efficient characterization of this new genomic variant, a full-length infectious genomic clone of HAdV-B14 was successfully constructed using one-step Gibson Assembly method in this study. Firstly, the full genome of HAdV-B14p1 strain GZ01, the first HAdV-B14 isolate in China, was assembled into pBR322 plasmid by Gibson Assembly. The pBRAdV14 plasmid, generated by Gibson Assembly, was analyzed and verified by PCR, restriction enzymes digestion and the sequencing. Secondly, viruses were rescued from pBRAdV14-transfected A549 cells. The integrity of the rescued viruses was identified by restriction enzyme analysis. The complete sequence of the infectious clone was further sequenced. No mutation was found in the infectious clone during the construction when compared with the parental virus and pBR322 sequences. The direct immunofluorescence assay indicated the expression of the hexon protein. Finally, typical virions were observed; the one-step growth curves further showed that the DNA replication and viral reproduction efficiency of pBRAd14 derived viruses was similar with that of wild-type HAdV-B14 strain. The successful construction of the replication-competent infectious clone of pBRAdV14 facilitates the development of vaccine and antiviral drugs against HAdV-B14, as well as provides a novel strategy for rapid construction of infectious viral clones for other large-genome DNA viruses.Viruses, Vol. 10, Pages 568: Rapid Construction of a Replication-Competent Infectious Clone of Human Adenovirus Type 14 by Gibson Assembly

In 1955, Human adenovirus type 14 (HAdV-B14p) was firstly identified in a military trainee diagnosed as acute respiratory disease (ARD) in the Netherlands. Fifty years later, a genomic variant, HAdV-B14p1, re-emerged in the U.S. and caused large and fatal ARD outbreaks. Subsequently, more and more ARD outbreaks occurred in Canada, the UK, Ireland, and China, in both military and civil settings. To generate a tool for the efficient characterization of this new genomic variant, a full-length infectious genomic clone of HAdV-B14 was successfully constructed using one-step Gibson Assembly method in this study. Firstly, the full genome of HAdV-B14p1 strain GZ01, the first HAdV-B14 isolate in China, was assembled into pBR322 plasmid by Gibson Assembly. The pBRAdV14 plasmid, generated by Gibson Assembly, was analyzed and verified by PCR, restriction enzymes digestion and the sequencing. Secondly, viruses were rescued from pBRAdV14-transfected A549 cells. The integrity of the rescued viruses was identified by restriction enzyme analysis. The complete sequence of the infectious clone was further sequenced. No mutation was found in the infectious clone during the construction when compared with the parental virus and pBR322 sequences. The direct immunofluorescence assay indicated the expression of the hexon protein. Finally, typical virions were observed; the one-step growth curves further showed that the DNA replication and viral reproduction efficiency of pBRAd14 derived viruses was similar with that of wild-type HAdV-B14 strain. The successful construction of the replication-competent infectious clone of pBRAdV14 facilitates the development of vaccine and antiviral drugs against HAdV-B14, as well as provides a novel strategy for rapid construction of infectious viral clones for other large-genome DNA viruses.

Viroids are smallest known pathogen that consist of non-capsidated, single-stranded non-coding RNA replicons and they exploits host factors for their replication and propagation. The severe stunting disease caused by Citrus bark cracking viroid (CBCVd) is a serious threat, which spreads rapidly within hop gardens. In this study, we employed comprehensive transcriptome analyses to dissect host-viroid interactions and identify gene expression changes that are associated with disease development in hop. Our analysis revealed that CBCVd-infection resulted in the massive modulation of activity of over 2000 genes. Expression of genes associated with plant immune responses (protein kinase and mitogen-activated protein kinase), hypersensitive responses, phytohormone signaling pathways, photosynthesis, pigment metabolism, protein metabolism, sugar metabolism, and modification, and others were altered, which could be attributed to systemic symptom development upon CBCVd-infection in hop. In addition, genes encoding RNA-dependent RNA polymerase, pathogenesis-related protein, chitinase, as well as those related to basal defense responses were up-regulated. The expression levels of several genes identified from RNA sequencing analysis were confirmed by qRT-PCR. Our systematic comprehensive CBCVd-responsive transcriptome analysis provides a better understanding and insights into complex viroid-hop plant interaction. This information will assist further in the development of future measures for the prevention of CBCVd spread in hop fields.

]]>Genome-Wide Transcriptomic Analysis Reveals Insights into the Response to Citrus bark cracking viroid (CBCVd) in Hop (Humulus lupulus L.)Ajay Kumar MishraAtul KumarDeepti MishraVishnu Sukumari NathJernej JakšeTomáš KocábekUday Kumar KilliFilis MorinaJaroslav Matoušekdoi: 10.3390/v10100570Viruses2018-10-18Viruses2018-10-181010Article57010.3390/v10100570http://www.mdpi.com/1999-4915/10/10/570Viruses, Vol. 10, Pages 569: Time to Harmonize Dengue Nomenclature and Classificationhttp://www.mdpi.com/1999-4915/10/10/569
Dengue virus (DENV) is estimated to cause 390 million infections per year worldwide. A quarter of these infections manifest clinically and are associated with a morbidity and mortality that put a significant burden on the affected regions. Reports of increased frequency, intensity, and extended geographical range of outbreaks highlight the virus&amp;rsquo;s ongoing global spread. Persistent transmission in endemic areas and the emergence in territories formerly devoid of transmission have shaped DENV&amp;rsquo;s current genetic diversity and divergence. This genetic layout is hierarchically organized in serotypes, genotypes, and sub-genotypic clades. While serotypes are well defined, the genotype nomenclature and classification system lack consistency, which complicates a broader analysis of their clinical and epidemiological characteristics. We identify five key challenges: (1) Currently, there is no formal definition of a DENV genotype; (2) Two different nomenclature systems are used in parallel, which causes significant confusion; (3) A standardized classification procedure is lacking so far; (4) No formal definition of sub-genotypic clades is in place; (5) There is no consensus on how to report antigenic diversity. Therefore, we believe that the time is right to re-evaluate DENV genetic diversity in an essential effort to provide harmonization across DENV studies.Viruses, Vol. 10, Pages 569: Time to Harmonize Dengue Nomenclature and Classification

Dengue virus (DENV) is estimated to cause 390 million infections per year worldwide. A quarter of these infections manifest clinically and are associated with a morbidity and mortality that put a significant burden on the affected regions. Reports of increased frequency, intensity, and extended geographical range of outbreaks highlight the virus&amp;rsquo;s ongoing global spread. Persistent transmission in endemic areas and the emergence in territories formerly devoid of transmission have shaped DENV&amp;rsquo;s current genetic diversity and divergence. This genetic layout is hierarchically organized in serotypes, genotypes, and sub-genotypic clades. While serotypes are well defined, the genotype nomenclature and classification system lack consistency, which complicates a broader analysis of their clinical and epidemiological characteristics. We identify five key challenges: (1) Currently, there is no formal definition of a DENV genotype; (2) Two different nomenclature systems are used in parallel, which causes significant confusion; (3) A standardized classification procedure is lacking so far; (4) No formal definition of sub-genotypic clades is in place; (5) There is no consensus on how to report antigenic diversity. Therefore, we believe that the time is right to re-evaluate DENV genetic diversity in an essential effort to provide harmonization across DENV studies.

]]>Time to Harmonize Dengue Nomenclature and ClassificationLize CuypersPieter J. K. LibinPeter SimmondsAnn NowéJorge Muñoz-JordánLuiz Carlos Junior AlcantaraAnne-Mieke VandammeGilberto A. SantiagoKristof Theysdoi: 10.3390/v10100569Viruses2018-10-18Viruses2018-10-181010Communication56910.3390/v10100569http://www.mdpi.com/1999-4915/10/10/569Viruses, Vol. 10, Pages 567: The Viral Tegument Protein pp65 Impairs Transcriptional Upregulation of IL-1β by Human Cytomegalovirus through Inhibition of NF-kB Activityhttp://www.mdpi.com/1999-4915/10/10/567
Interleukin-1&amp;beta; (IL-1&amp;beta;) is a key effector of the inflammasome complex in response to pathogens and danger signals. Although it is well known that assembly of the inflammasome triggers proteolytic cleavage of the biologically inactive precursor pro-IL-1&amp;beta; into its mature secreted form, the mechanism by which human cytomegalovirus (HCMV) regulates IL-1&amp;beta; production via the inflammasome is still poorly understood. Here, we show that the infection of human foreskin fibroblasts (HFFs) with a mutant HCMV lacking the tegument protein pp65 (v65Stop) results in higher expression levels of mature IL-1&amp;beta; compared to its wild-type counterpart, suggesting that pp65 mediates HCMV immune evasion through downmodulation of IL-1&amp;beta;. Furthermore, we show that enhanced IL-1&amp;beta; production by the v65Stop mutant is due in part to induction of DNA binding and the transcriptional activity of NF-&amp;kappa;B. Lastly, we demonstrate that HCMV infection of HFFs triggers a non-canonical IL-1&amp;beta; activation pathway where caspase-8 promotes IL-1&amp;beta; maturation independently of caspase-1. Altogether, our findings provide novel mechanistic insights into the interplay between HCMV and the inflammasome system and raise the possibility of targeting pp65 to treat HCMV infection.Viruses, Vol. 10, Pages 567: The Viral Tegument Protein pp65 Impairs Transcriptional Upregulation of IL-1β by Human Cytomegalovirus through Inhibition of NF-kB Activity

Interleukin-1&amp;beta; (IL-1&amp;beta;) is a key effector of the inflammasome complex in response to pathogens and danger signals. Although it is well known that assembly of the inflammasome triggers proteolytic cleavage of the biologically inactive precursor pro-IL-1&amp;beta; into its mature secreted form, the mechanism by which human cytomegalovirus (HCMV) regulates IL-1&amp;beta; production via the inflammasome is still poorly understood. Here, we show that the infection of human foreskin fibroblasts (HFFs) with a mutant HCMV lacking the tegument protein pp65 (v65Stop) results in higher expression levels of mature IL-1&amp;beta; compared to its wild-type counterpart, suggesting that pp65 mediates HCMV immune evasion through downmodulation of IL-1&amp;beta;. Furthermore, we show that enhanced IL-1&amp;beta; production by the v65Stop mutant is due in part to induction of DNA binding and the transcriptional activity of NF-&amp;kappa;B. Lastly, we demonstrate that HCMV infection of HFFs triggers a non-canonical IL-1&amp;beta; activation pathway where caspase-8 promotes IL-1&amp;beta; maturation independently of caspase-1. Altogether, our findings provide novel mechanistic insights into the interplay between HCMV and the inflammasome system and raise the possibility of targeting pp65 to treat HCMV infection.

]]>The Viral Tegument Protein pp65 Impairs Transcriptional Upregulation of IL-1β by Human Cytomegalovirus through Inhibition of NF-kB ActivityMatteo BiolattiValentina Dell’OsteSara ScuteraFrancesca GugliesiGloria GriffanteMarco De AndreaTiziana MussoSanto Landolfodoi: 10.3390/v10100567Viruses2018-10-16Viruses2018-10-161010Communication56710.3390/v10100567http://www.mdpi.com/1999-4915/10/10/567Viruses, Vol. 10, Pages 564: Saccharomyces paradoxus K66 Killer System Evidences Expanded Assortment of Helper and Satellite Viruseshttp://www.mdpi.com/1999-4915/10/10/564
The Saccharomycetaceae yeast family recently became recognized for expanding of the repertoire of different dsRNA-based viruses, highlighting the need for understanding of their cross-dependence. We isolated the Saccharomyces paradoxus AML-15-66 killer strain from spontaneous fermentation of serviceberries and identified helper and satellite viruses of the family Totiviridae, which are responsible for the killing phenotype. The corresponding full dsRNA genomes of viruses have been cloned and sequenced. Sequence analysis of SpV-LA-66 identified it to be most similar to S. paradoxus LA-28 type viruses, while SpV-M66 was mostly similar to the SpV-M21 virus. Sequence and functional analysis revealed significant differences between the K66 and the K28 toxins. The structural organization of the K66 protein resembled those of the K1/K2 type toxins. The AML-15-66 strain possesses the most expressed killing property towards the K28 toxin-producing strain. A genetic screen performed on S. cerevisiae YKO library strains revealed 125 gene products important for the functioning of the S. paradoxus K66 toxin, with 85% of the discovered modulators shared with S. cerevisiae K2 or K1 toxins. Investigation of the K66 protein binding to cells and different polysaccharides implies the &amp;beta;-1,6 glucans to be the primary receptors of S. paradoxus K66 toxin. For the first time, we demonstrated the coherent habitation of different types of helper and satellite viruses in a wild-type S. paradoxus strain.Viruses, Vol. 10, Pages 564: Saccharomyces paradoxus K66 Killer System Evidences Expanded Assortment of Helper and Satellite Viruses

The Saccharomycetaceae yeast family recently became recognized for expanding of the repertoire of different dsRNA-based viruses, highlighting the need for understanding of their cross-dependence. We isolated the Saccharomyces paradoxus AML-15-66 killer strain from spontaneous fermentation of serviceberries and identified helper and satellite viruses of the family Totiviridae, which are responsible for the killing phenotype. The corresponding full dsRNA genomes of viruses have been cloned and sequenced. Sequence analysis of SpV-LA-66 identified it to be most similar to S. paradoxus LA-28 type viruses, while SpV-M66 was mostly similar to the SpV-M21 virus. Sequence and functional analysis revealed significant differences between the K66 and the K28 toxins. The structural organization of the K66 protein resembled those of the K1/K2 type toxins. The AML-15-66 strain possesses the most expressed killing property towards the K28 toxin-producing strain. A genetic screen performed on S. cerevisiae YKO library strains revealed 125 gene products important for the functioning of the S. paradoxus K66 toxin, with 85% of the discovered modulators shared with S. cerevisiae K2 or K1 toxins. Investigation of the K66 protein binding to cells and different polysaccharides implies the &amp;beta;-1,6 glucans to be the primary receptors of S. paradoxus K66 toxin. For the first time, we demonstrated the coherent habitation of different types of helper and satellite viruses in a wild-type S. paradoxus strain.

]]>Saccharomyces paradoxus K66 Killer System Evidences Expanded Assortment of Helper and Satellite VirusesIglė Vepštaitė-MonstavičėJuliana LukšaAleksandras KonovalovasDovilė EžerskytėRamunė StanevičienėŽivilė Strazdaitė-ŽielienėSaulius ServaElena Servienėdoi: 10.3390/v10100564Viruses2018-10-16Viruses2018-10-161010Article56410.3390/v10100564http://www.mdpi.com/1999-4915/10/10/564Viruses, Vol. 10, Pages 566: Current Perspectives on High-Throughput Sequencing (HTS) for Adventitious Virus Detection: Upstream Sample Processing and Library Preparationhttp://www.mdpi.com/1999-4915/10/10/566
A key step for broad viral detection using high-throughput sequencing (HTS) is optimizing the sample preparation strategy for extracting viral-specific nucleic acids since viral genomes are diverse: They can be single-stranded or double-stranded RNA or DNA, and can vary from a few thousand bases to over millions of bases, which might introduce biases during nucleic acid extraction. In addition, viral particles can be enveloped or non-enveloped with variable resistance to pre-treatment, which may influence their susceptibility to extraction procedures. Since the identity of the potential adventitious agents is unknown prior to their detection, efficient sample preparation should be unbiased toward all different viral types in order to maximize the probability of detecting any potential adventitious viruses using HTS. Furthermore, the quality assessment of each step for sample processing is also a critical but challenging aspect. This paper presents our current perspectives for optimizing upstream sample processing and library preparation as part of the discussion in the Advanced Virus Detection Technologies Interest group (AVDTIG). The topics include: Use of nuclease treatment to enrich for encapsidated nucleic acids, techniques for amplifying low amounts of virus nucleic acids, selection of different extraction methods, relevant controls, the use of spike recovery experiments, and quality control measures during library preparation.Viruses, Vol. 10, Pages 566: Current Perspectives on High-Throughput Sequencing (HTS) for Adventitious Virus Detection: Upstream Sample Processing and Library Preparation

A key step for broad viral detection using high-throughput sequencing (HTS) is optimizing the sample preparation strategy for extracting viral-specific nucleic acids since viral genomes are diverse: They can be single-stranded or double-stranded RNA or DNA, and can vary from a few thousand bases to over millions of bases, which might introduce biases during nucleic acid extraction. In addition, viral particles can be enveloped or non-enveloped with variable resistance to pre-treatment, which may influence their susceptibility to extraction procedures. Since the identity of the potential adventitious agents is unknown prior to their detection, efficient sample preparation should be unbiased toward all different viral types in order to maximize the probability of detecting any potential adventitious viruses using HTS. Furthermore, the quality assessment of each step for sample processing is also a critical but challenging aspect. This paper presents our current perspectives for optimizing upstream sample processing and library preparation as part of the discussion in the Advanced Virus Detection Technologies Interest group (AVDTIG). The topics include: Use of nuclease treatment to enrich for encapsidated nucleic acids, techniques for amplifying low amounts of virus nucleic acids, selection of different extraction methods, relevant controls, the use of spike recovery experiments, and quality control measures during library preparation.

]]>Current Perspectives on High-Throughput Sequencing (HTS) for Adventitious Virus Detection: Upstream Sample Processing and Library PreparationSiemon NgCassandra BraxtonMarc EloitSzi FengRomain FragnoudLaurent MalletEdward MeeSarmitha SathiamoorthyOlivier VandeputteArifa Khandoi: 10.3390/v10100566Viruses2018-10-16Viruses2018-10-161010Perspective56610.3390/v10100566http://www.mdpi.com/1999-4915/10/10/566Viruses, Vol. 10, Pages 565: Canine Influenza Virus is Mildly Restricted by Canine Tetherin Proteinhttp://www.mdpi.com/1999-4915/10/10/565
Tetherin (BST2/CD317/HM1.24) has emerged as a key host-cell &amp;middot;defence molecule that acts by inhibiting the release and spread of diverse enveloped virions from infected cells. We analysed the biological features of canine tetherin and found it to be an unstable hydrophilic type I transmembrane protein with one transmembrane domain, no signal peptide, and multiple glycosylation and phosphorylation sites. Furthermore, the tissue expression profile of canine tetherin revealed that it was particularly abundant in immune organs. The canine tetherin gene contains an interferon response element sequence that can be regulated and expressed by canine IFN-&amp;alpha;. A CCK-8 assay showed that canine tetherin was effective in helping mitigate cellular damage caused by canine influenza virus (CIV) infection. Additionally, we found that the overexpression of canine tetherin inhibited replication of the CIV and that interference with the canine tetherin gene enhanced CIV replication in cells. The impact of canine tetherin on CIV replication was mild. However, these results elucidate the role of the innate immune factor, canine tetherin, during CIV infection for the first time.Viruses, Vol. 10, Pages 565: Canine Influenza Virus is Mildly Restricted by Canine Tetherin Protein

Tetherin (BST2/CD317/HM1.24) has emerged as a key host-cell &amp;middot;defence molecule that acts by inhibiting the release and spread of diverse enveloped virions from infected cells. We analysed the biological features of canine tetherin and found it to be an unstable hydrophilic type I transmembrane protein with one transmembrane domain, no signal peptide, and multiple glycosylation and phosphorylation sites. Furthermore, the tissue expression profile of canine tetherin revealed that it was particularly abundant in immune organs. The canine tetherin gene contains an interferon response element sequence that can be regulated and expressed by canine IFN-&amp;alpha;. A CCK-8 assay showed that canine tetherin was effective in helping mitigate cellular damage caused by canine influenza virus (CIV) infection. Additionally, we found that the overexpression of canine tetherin inhibited replication of the CIV and that interference with the canine tetherin gene enhanced CIV replication in cells. The impact of canine tetherin on CIV replication was mild. However, these results elucidate the role of the innate immune factor, canine tetherin, during CIV infection for the first time.

Members of the family Filoviridae, including Ebola virus (EBOV) and Marburg virus (MARV), cause severe hemorrhagic fever in humans and nonhuman primates. Given their high lethality, a comprehensive understanding of filoviral pathogenesis is urgently needed. In the present studies, we revealed that the exchange protein directly activated by cAMP 1 (EPAC1) gene deletion protects vasculature in ex vivo explants from EBOV infection. Importantly, pharmacological inhibition of EPAC1 using EPAC-specific inhibitors (ESIs) mimicked the EPAC1 knockout phenotype in the ex vivo model. ESI treatment dramatically decreased EBOV infectivity in both ex vivo vasculature and in vitro vascular endothelial cells (ECs). Furthermore, postexposure protection of ECs against EBOV infection was conferred using ESIs. Protective efficacy of ESIs in ECs was observed also in MARV infection. Additional studies using a vesicular stomatitis virus pseudotype that expresses EBOV glycoprotein (EGP-VSV) confirmed that ESIs reduced infection in ECs. Ultrastructural studies suggested that ESIs blocked EGP-VSV internalization via inhibition of macropinocytosis. The inactivation of EPAC1 affects the early stage of viral entry after viral binding to the cell surface, but before early endosome formation, in a phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)-dependent manner. Our study delineated a new critical role of EPAC1 during EBOV uptake into ECs.